Selection of patients for combination therapy

ABSTRACT

Described herein are methods for selecting cancer patients for treatment with a combination therapy comprising entinostat and a second therapeutic agent. In particular, methods are provided for the examination of a non-cancer cell type, myeloid-derived suppressor cells, e.g., those which are CD14-positive and HLA-DR-(lo/negative), as a therapeutic indicator in the setting of entinostat combination therapies.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/756,898, filed Mar. 1, 2018, which is a national stage application ofand claims priority to International Application No. PCT/US2016/050274filed Sep. 2, 2016, which in turn claims the benefit of and priority toU.S. Provisional Application Nos. 62/213,288, filed Sep. 2, 2015, and62/219,612, filed Sep. 16, 2015, the contents of each of which arehereby incorporated by reference in their entireties.

BACKGROUND

Cancer, tumors, tumor-related disorders, and neoplastic disease statesare serious and often times life-threatening conditions. These diseasesand disorders, which are characterized by rapidly-proliferating cellgrowth, continue to be the subject of research efforts directed towardthe identification of therapeutic agents which are effective in thetreatment thereof. Such agents prolong the survival of the patient,inhibit the rapidly-proliferating cell growth associated with theneoplasm, or effect a regression of the neoplasm.

HDAC inhibitors (HDACi) are an emerging class of therapeutic agents thatpromote differentiation and apoptosis in hematologic and solidmalignancies through chromatin remodeling and gene expressionregulation. Although the antitumor effects of HDACi have been studied,the impact of HDACi on cancer patient systemic immunity remains unclear.

There is a need for cancer immunotherapy in multiple indications, e.g.,to increase the effectiveness of the antitumor agents and reduce and/oreliminate the side effects typically associated with conventionaltreatment.

SUMMARY

In one aspect, provided herein is a method of selecting a patient forcombination therapy comprising entinostat and a second therapeuticagent. The method comprises obtaining a peripheral blood sample from thepatient, wherein the patient is diagnosed with a cancer, measuring thenumber of myeloid derived suppressor cells and peripheral bloodmononuclear cells in the peripheral blood sample; and administering thecombination therapy if the ratio of myeloid derived suppressor cells toperipheral blood mononuclear cells is between 1:200 and 1:4.

In another aspect, provided herein is a method of selecting a patientfor combination therapy comprising entinostat and a second therapeuticagent. The method comprises obtaining a peripheral blood sample from thepatient, wherein the patient is diagnosed with a cancer, contacting oneor more myeloid derived suppressor cells from the peripheral bloodsample with a first binding agent to generate one or more first bindingagent-myeloid derived suppressor cell complexes, contacting one or moreperipheral blood mononuclear cells from the peripheral blood sample witha second binding agent to generate one or more second bindingagent-peripheral blood mononuclear cell complexes, measuring a ratio ofthe first binding agent-myeloid derived suppressor cell complexes andthe second binding agent-peripheral blood mononuclear cell complexes inthe peripheral blood sample, and administering the combination therapyif the ratio of first binding agent-myeloid derived suppressor cellcomplexes to second binding agent-peripheral blood mononuclear cellcomplexes is between 1:200 and 1:4.

In another aspect, provided herein is a method of providing a prognosisfor cancer in a patient comprising obtaining a peripheral blood samplefrom the patient, wherein the patient is diagnosed with a cancer,measuring the number of myeloid derived suppressor cells and peripheralblood mononuclear cells in the peripheral blood sample, wherein themethod further comprises, administering a combination therapy comprisingentinostat and a second therapeutic agent to the patient, if the ratioof myeloid derived suppressor cells to peripheral blood mononuclearcells is between 1:200 and 1:4.

In yet another aspect, provided herein is a method of selecting apatient for combination therapy comprising entinostat and a secondtherapeutic agent comprising: obtaining a peripheral blood sample fromthe patient, wherein the patient is diagnosed with a cancer; measuringthe number of cells in the peripheral blood sample which areCD14-positive and HLA-DR-(lo/negative); measuring the number of cells inthe peripheral blood sample which are CD-14 positive; and administeringthe combination therapy if the ratio of the CD14-positive andHLA-DR-(lo/negative) cells to CD-14 positive cells is between 1:100 and99:1.

Provided in another aspect is a method of selecting a patient forcombination therapy comprising entinostat and a second therapeutic agentcomprising: obtaining a peripheral blood sample from the patient,wherein the patient is diagnosed with a cancer; measuring the number ofcells in the peripheral blood sample which are CD14-positive andHLA-DR-(lo/negative); measuring the number peripheral blood mononuclearcells in the peripheral blood sample; and

administering the combination therapy if the ratio of the CD14-positiveand HLA-DR-(lo/negative) cells to peripheral blood mononuclear cells isbetween 1:200 and 1:1.

In still another aspect, provided herein is a method of providing aprognosis for cancer in a patient comprising: obtaining a peripheralblood sample from the patient, wherein the patient is diagnosed with acancer; measuring the number of cells in the peripheral blood samplewhich are CD14-positive and HLA-DR-(lo/negative); measuring the numberof cells in the peripheral blood sample which are CD-14 positive,wherein the method further comprises, administering a combinationtherapy comprising entinostat and a second therapeutic agent to thepatient, if the ratio of CD14-positive and HLA-DR-(lo/negative) cells toCD-14 positive cells is between 1:100 and 99:1.

Also provided herein is a method of providing a prognosis for cancer ina patient comprising: obtaining a peripheral blood sample from thepatient, wherein the patient is diagnosed with a cancer; measuring thenumber of cells in the peripheral blood sample which are CD14-positiveand HLA-DR-(lo/negative); measuring the number of peripheral bloodmononuclear cells in the peripheral blood sample, wherein the methodfurther comprises, administering a combination therapy comprisingentinostat and a second therapeutic agent to the patient, if the ratioof CD14-positive and HLA-DR-(lo/negative) cells to peripheral bloodmononuclear cells is between 1:200 and 1:1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates percentage change of CD14+ cell number,CD14+HLA-DR^(Hi) monocyte cell number, monocytic MDSCs cell number, CD40expression on monocytic MDSCs, granulocytic MDSCs cell number, and CD40expression on monocytic MDSCs, in blood samples obtained from selectedpatients from ENCORE 301 trial, between baseline to day 15 of treatmentwith either a combination of entinostat and exemestane (EE) or acombination of exemestane and placebo.

FIG. 2 illustrates that entinostat increases HLA-DR expression on CD14⁺monocytes in breast cancer patients. (A) Gating strategy for analysis ofCD14⁺ monocytes (left panel), CD14⁺HLA-DR^(hi) monocytes (red box, rightupper panel), and CD14⁺HLA-DR^(low/neg) monocytes (blue box, right lowerpanel) in PBMCs of breast cancer patients. Initially gated on singleviable CD45⁺ cells. (B) Change of % CD14⁺HLA-DRhi monocytes among singleviable CD45⁺ PBMCs from baseline to C1D15 in exemestane plus placebo(EP) arm (n=14) and exemestane plus entinostat (EE) arm (n=20). Thelevel of CD14⁺HLA-DRhi monocytes was significantly increased in the EEarm compared to the EP arm (P=0.0004). (C) Change of HLA-DR expression(median fluorescence intensity, MFI) on CD14⁺ monocytes from baseline toC1D15 in the EP arm (n=14) and EE arm (n=20). The level of HLA-DRexpression on CD14⁺ monocytes was significantly increased in the EE armcompared to the EP arm (P=0.015). (D) HLA-DR expression on CD14⁺monocytes in vitro. Fresh PBMCs were cultured with DMSO or entinostat(0.5 μM) for two days. Left panel shows a representative histogram ofHLA-DR expression on CD14⁺ monocytes cultured with DMSO (blue histogram)or with entinostat (red histogram). Black histogram shows isotypecontrol. Right panel shows the difference of HLA-DR expression levels onCD14⁺ monocytes cultured with DMSO or entinostat. Each line represents adifferent healthy donor (n=8, P=0.008). Median fluorescence intensity,MFI.

FIG. 3 illustrates that entinostat decreases monocytic MDSCs andgranulocytic MDSCs in breast cancer patients. (A) Gating strategy foranalysis of MDSC phenotypes in PBMCs of breast cancer patients. Initialgating was on single viable CD45⁺ cells. Lineage (CD3, CD19,CD56)⁻HLA-DR⁻CD11b⁺CD33⁺ cells were defined as Lin⁻MDSCs. The Lin⁻MDSCswere further divided into monocytic MDSCs (Lin⁻HLA-DR⁻CD11b⁺CD33⁺CD14⁺cells) and immature MDSCs (Lin⁻HLA-DR⁻CD11b⁺CD33⁺CD14⁻ cells).CD14⁻CD11b⁺CD33⁺ cells were defined as granulocytic MDSCs. (B) Change of% monocytic MDSCs among single viable CD45⁺ PBMCs from baseline to C1D15in the exemestane plus placebo (EP) arm (n=14) and the exemestane plusentinostat (EE) arm (n=20). The level of monocytic MDSCs wassignificantly decreased in the EE arm compared to the EP arm (P=0.002).(C) Change of % granulocytic MDSCs among single viable CD45⁺ PBMCs frombaseline to C1D15 in the EP (n=14) and the EE arm (n=20). The level ofgranulocytic MDSCs was significantly decreased in the EE arm compared tothe EP arm (P=0.029).

FIG. 4 illustrates that entinostat decreases CD40 expression on MDSCs inbreast cancer patients. (A) Change of CD40 expression (MFI) on monocyticMDSCs from baseline to C1D15 in exemestane plus placebo (EP) arm (n=14)and exemestane plus entinostat (EE) arm (n=20). The level of CD40 onmonocytic MDSCs was significantly decreased in the EE arm compared tothe EP arm (P =0.011). (B) Change of CD40 expression (MFI) ongranulocytic MDSCs from baseline to C1D15 in the EP arm (n=14) and theEE arm (n=20). The level of CD40 on granulocytic MDSCs did not show astatistically significant decrease in the EE arm compared to the EP arm(P=0.22). (C) Absolute viable cell counts of MDSCs (upper panel,monocytic MDSCs; lower panel, granulocytic MDSCs). Fresh PBMCs (2×10⁶PBMCs/well) were cultured with IL-6 (10 ng/ml) and GM-CSF (10 ng/ml). Onday 5, DMSO or entinostat (0.5 μM) was added and cells were cultured for3-4 days. Each line represents a different healthy donor (n=7; monocyticMDSCs, P=0.004; granulocytic MDSCs, P=0.004). (D) Percentage of deadcell dye-positive cells in the monocytic MDSCs (red line), granulocyticMDSCs (blue line), and Lineage cells (black line). Fresh PBMCs (2×10PBMCs/well) were cultured with IL-6 and GM-CSF and then, DMSO orentinostat (0.5 μM) was added for two or three days and then cells werecollected and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain andantibodies. Percentage of dead cell dye-positive cells among eachpopulation (monocytic MDSCs, granulocytic MDSCs, and lineage cells) wascalculated. Mean±SD is shown (n=7). Percentage of dead monocytic MDSCsand dead granulocytic MDSCs was increased by entinostat although thepercentage of dead lineage cells was not increased. Monocytic MDSCs,P=0.016; Granulocytic MDSCs, P=0.016; Lineage cells (CD3⁺, CD19⁺, orCD56⁺), P=0.7.

FIG. 5 illustrates gating strategy for analysis of T-cell subsets inPBMCs of breast cancer patients. (A) Initial gating was on single viablecells. The CD4⁺ T-cells were further divided into Tregs(CD8⁻CD4⁺CD25^(hi) Foxp3⁺ cells) and Foxp3⁻CD4⁺ T-cells (CD8⁻CD4⁺Foxp3⁻cells). (B) Immune checkpoint receptor expression was evaluated forCD8⁺T-cells, Foxp3⁻CD4⁺ T-cells, and Tregs. Representative histogramsfor PD-1 (left), CTLA-4 (middle), and TIM-3 (right) were shown.

DETAILED DESCRIPTION

Conventional approaches for selecting cancer patients for combinationtherapy rely upon assessment of the cancer, either in terms of histologyor molecular analyses. The present disclosure provides methods that relyupon the levels of non-tumor myeloid derived suppressor cells, e.g.,those which are CD14-positive and HLA-DR-(lo/negative), in a biologicalsample obtained from a cancer patient, as predictive and prognosticbiomarker for selecting a patient for a combination therapy withentinostat and a second therapeutic agent.

In one aspect, the disclosure relates to a method of selecting a patientfor combination therapy comprising entinostat and a second therapeuticagent, based on the ratio of myeloid derived suppressor cells andperipheral blood mononuclear cells, measured in peripheral blood samplecollected from the patients, wherein the combination therapy isadministered if the ratio of myeloid derived suppressor cells toperipheral blood mononuclear cells is between 1:200 and 1:4. Providedherein in another aspect is a method of selecting a patient forcombination therapy comprising entinostat and a second therapeuticagent, based on the ratio of myeloid derived suppressor cells-firstbinding agent complexes and peripheral blood mononuclear cells-secondbinding agent complexes, measured in peripheral blood sample collectedfrom the patients, wherein the complexes are formed by contacting themyeloid derived suppressor cells and the peripheral blood mononuclearcells with the first and second bindings respectively, and wherein thecombination therapy is administered if the ratio of myeloid derivedsuppressor cell first binding agent complexes to peripheral bloodmononuclear cell-second binding agent complexes is between 1:200 and1:4. Further provided in another aspect is a method for providing adiagnosis and/or prognosis of cancer in a patient based on the ratio ofmyeloid derived suppressor cells and peripheral blood mononuclear cells,measured in peripheral blood sample collected from the patients, whereinthe method further comprises administering a combination therapycomprising entinostat and a second therapeutic agent if the ratio ofmyeloid derived suppressor cells to peripheral blood mononuclear cellsis between 1:200 and 1:4.

In yet another aspect, provided herein is a method of selecting apatient for combination therapy comprising entinostat and a secondtherapeutic agent comprising: obtaining a peripheral blood sample fromthe patient, wherein the patient is diagnosed with a cancer; measuringthe number of cells in the peripheral blood sample which areCD14-positive and HLA-DR-(lo/negative); measuring the number of cells inthe peripheral blood sample which are CD-14 positive; and administeringthe combination therapy if the ratio of the CD14-positive andHLA-DR-(lo/negative) cells to CD-14 positive cells is between 1:100 and99:1. Provided in another aspect is a method of selecting a patient forcombination therapy comprising entinostat and a second therapeutic agentcomprising: obtaining a peripheral blood sample from the patient,wherein the patient is diagnosed with a cancer; measuring the number ofcells in the peripheral blood sample which are CD14-positive andHLA-DR-(lo/negative); measuring the number peripheral blood mononuclearcells in the peripheral blood sample; and administering the combinationtherapy if the ratio of the CD14-positive and HLA-DR-(lo/negative) cellsto peripheral blood mononuclear cells is between 1:200 and 1:1. Providedherein in yet another aspect is a method of providing a prognosis forcancer in a patient comprising: obtaining a peripheral blood sample fromthe patient, wherein the patient is diagnosed with a cancer; measuringthe number of cells in the peripheral blood sample which areCD14-positive and HLA-DR-(lo/negative); measuring the number of cells inthe peripheral blood sample which are CD-14 positive, wherein the methodfurther comprises, administering a combination therapy comprisingentinostat and a second therapeutic agent to the patient, if the ratioof CD14-positive and HLA-DR-(lo/negative) cells to CD-14 positive cellsis between 1:100 and 99:1. Provided herein in another aspect, is amethod of providing a prognosis for cancer in a patient comprising:obtaining a peripheral blood sample from the patient, wherein thepatient is diagnosed with a cancer; measuring the number of cells in theperipheral blood sample which are CD14-positive andHLA-DR-(lo/negative); measuring the number of peripheral bloodmononuclear cells in the peripheral blood sample, wherein the methodfurther comprises, administering a combination therapy comprisingentinostat and a second therapeutic agent to the patient, if the ratioof CD14-positive and HLA-DR-(lo/negative) cells to peripheral bloodmononuclear cells is between 1:200 and 1:1.

In some embodiments, the peripheral blood sample is treated with ananticoagulant. In some embodiments, the anticoagulant is EDTA orheparin. In some embodiments, measuring number of cells in theperipheral blood sample which are CD14-positive and HLA-DR-(lo/negative)is performed by flow cytometry. In some embodiments, the peripheralblood mononuclear cell population is identified by a cell surfacemarker. In some embodiments, the cell surface marker is at least one ofCD3, CD14, CD19, CD56, and HLA-DR. In some embodiments, the ratio of theCD14-positive and HLA-DR-(lo/negative) cells to CD-14 positive cells isbetween 1:50 and 99:1. In some embodiments, the ratio the ratio of theCD14-positive and HLA-DR-(lo/negative) cells to CD-14 positive cells isbetween 1:20 and 99:1. In some embodiments, the ratio of theCD14-positive and HLA-DR-(lo/negative) cells to CD-14 positive cells isbetween 1:10 and 99:1. In some embodiments, the ratio of theCD14-positive and HLA-DR-(lo/negative) cells to peripheral bloodmononuclear cells is between 1:5 and 99:1. In some embodiments, theratio of the CD14-positive and HLA-DR-(lo/negative) cells to peripheralblood mononuclear cells is between 1:50 and 1:4. In some embodiments,the ratio the ratio of the CD14-positive and HLA-DR-(lo/negative) cellsto peripheral blood mononuclear cells is between 1:20 and 1:4. In someembodiments, the ratio of the CD14-positive and HLA-DR-(lo/negative)cells to peripheral blood mononuclear cells is between 1:10 and 1:4. Insome embodiments, the ratio of the CD14-positive andHLA-DR-(lo/negative) cells to peripheral blood mononuclear cells isbetween 1:5 and 1:4.

In some embodiments, the entinostat is administered orally. In someembodiments, the entinostat is administered first. In some embodiments,the entinostat is administered weekly. In some embodiments, theentinostat is administered every two weeks. In some embodiments, theentinostat is administered at a dose of 5 mg. In some embodiments, theentinostat is administered at a dose of 5 mg weekly. In someembodiments, the entinostat is administered at a dose of 5 mg every twoweeks.

In some embodiments, the second therapeutic agent is an anti-PD-1antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab.In some embodiments, the anti-PD-1 antibody is nivolumab. In someembodiments, the cancer is a lung cancer. In some embodiments, the lungcancer is a non-small cell lung cancer, squamous cell carcinoma, orlarge cell carcinoma. In some embodiments, the cancer is a melanoma. Insome embodiments, the melanoma is a metastatic melanoma. In someembodiments, the second therapeutic agent is an anti-PD-L1 antibody. Insome embodiments, the anti-PD-L1 antibody is MPDL3280A. In someembodiments, the second therapeutic agent is exemestane. In someembodiments, the cancer is a breast cancer. In some embodiments, thesecond therapeutic agent is MPDL3280A and the breast cancer is atriple-negative breast cancer. In some embodiments, the secondtherapeutic agent is exemestane and the breast cancer is hormonereceptor positive breast cancer. In some embodiments, the anti-PD-1antibody or anti-PD-L1 antibody is administered by infusion. In someembodiments, the exemestane is administered orally.

To facilitate understanding of the disclosure set forth herein, a numberof terms are defined below.

As used herein, “abnormal cell growth,” refers to cell growth that isindependent of normal regulatory mechanisms (e.g., loss of contactinhibition), including the abnormal growth of normal cells and thegrowth of abnormal cells.

“Neoplasia” as described herein, is an abnormal, unregulated anddisorganized proliferation of cells that is distinguished from normalcells by autonomous growth and somatic mutations. As neoplastic cellsgrow and divide they pass on their genetic mutations and proliferativecharacteristics to progeny cells. A neoplasm, or tumor, is anaccumulation of neoplastic cells. In some embodiments, the neoplasm canbe benign or malignant.

“Metastasis,” as used herein, refers to the dissemination of tumor cellsvia lymphatics or blood vessels. Metastasis also refers to the migrationof tumor cells by direct extension through serous cavities, orsubarachnoid or other spaces. Through the process of metastasis, tumorcell migration to other areas of the body establishes neoplasms in areasaway from the site of initial appearance.

As discussed herein, “angiogenesis” is prominent in tumor formation andmetastasis. Angiogenic factors have been found associated with severalsolid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, and osteosarcoma. A tumor cannot expand without a bloodsupply to provide nutrients and remove cellular wastes. Tumors in whichangiogenesis is important include solid tumors such as renal cellcarcinoma, hepatocellular carcinoma, and benign tumors such as acousticneuroma, and neurofibroma. Angiogenesis has been associated withblood-born tumors such as leukemias. It is believed that angiogenesisplays a role in the abnormalities in the bone marrow that give rise toleukemia. Prevention of angiogenesis could halt the growth of canceroustumors and the resultant damage to the subject due to the presence ofthe tumor.

The term “subject” refers to an animal, including, but not limited to, aprimate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat,or mouse. The terms “subject” and “patient” are used interchangeablyherein in reference, for example, to a mammalian subject, such as ahuman subject.

The terms “treat,” “treating,” and “treatment” are meant to includealleviating or abrogating a disorder, disease, or condition; or one ormore of the symptoms associated with the disorder, disease, orcondition; or alleviating or eradicating the cause(s) of the disorder,disease, or condition itself.

The term “therapeutically effective amount” refers to the amount of acompound that, when administered, is sufficient to prevent developmentof, or alleviate to some extent, one or more of the symptoms of thedisorder, disease, or condition being treated. The term “therapeuticallyeffective amount” also refers to the amount of a compound that issufficient to elicit the biological or medical response of a cell,tissue, system, animal, or human that is being sought by a researcher,veterinarian, medical doctor, or clinician.

The term “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable excipient,” “physiologically acceptable carrier,” or“physiologically acceptable excipient” refers to apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent, or encapsulatingmaterial. Each component must be “pharmaceutically acceptable” in thesense of being compatible with the other ingredients of a pharmaceuticalformulation. It must also be suitable for use in contact with the tissueor organ of humans and animals without excessive toxicity, irritation,allergic response, immunogenicity, or other problems or complications,commensurate with a reasonable benefit/risk ratio. See, Remington: TheScience and Practice of Pharmacy, 21st Edition; Lippincott Williams &Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients,5th Edition; Rowe et al., Eds., The Pharmaceutical Press and theAmerican Pharmaceutical Association: 2005; and Handbook ofPharmaceutical Additives, 3rd Edition; Ash and Ash Eds., GowerPublishing Company: 2007; Pharmaceutical Preformulation and Formulation,Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004).

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.Pharmaceutical compositions can also be obtained by reacting compoundswith inorganic or organic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

The term “lo/negative”, when used to describe expression of a cellsurface marker on a cell, corresponds to a low to absent level ofexpression of the cell surface marker on the cell relative to anunstained control cell. A low to absent level of expression can be abouta 1-fold increase, about a 2-fold increase, about a 3-fold increase,about a 4-fold increase, about a 5-fold increase, about a 6-foldincrease, about a 7-fold increase, about a 8-fold increase, about a9-fold increase, about a 10-fold increase, can be about a 11-foldincrease, about a 12-fold increase, about a 13-fold increase, about a14-fold increase, about a 15-fold increase, about a 16-fold increase,about a 17-fold increase, about a 18-fold increase, about a 19-foldincrease, or about a 20-fold increase relative to an unstained controlcell. A low to absent level of expression can be from about a 1-foldincrease to about a 2-fold increase, from about a 2-fold increase toabout a 3-fold increase, from about a 3-fold increase to about a 4-foldincrease, from about a 4-fold increase to about a 5-fold increase, fromabout a 5-fold increase to about a 6-fold increase, from about a 6-foldincrease to about a 7-fold increase, from about a 7-fold increase toabout a 8-fold increase, from about a 8-fold increase to about a 9-foldincrease, from about a 9-fold increase to about a 10-fold increase, fromabout a 10-fold increase to about a 11-fold increase, from about a11-fold increase to about a 12-fold increase, from about a 12-foldincrease to about a 13-fold increase, from about a 13-fold increase toabout a 14-fold increase, from about a 14-fold increase to about a15-fold increase, from about a 15-fold increase to about a 16-foldincrease, from about a 16-fold increase to about a 17-fold increase,from about a 17-fold increase to about a 18-fold increase, from about a18-fold increase to about a 19-fold increase, or from about a 19-foldincrease to about a 20-fold increase.

Cancer, tumors, tumor-related disorders, and neoplastic disease statesare serious and often times life-threatening conditions. These diseasesand disorders, which are characterized by rapidly-proliferating cellgrowth, continue to be the subject of research efforts directed towardthe identification of therapeutic agents which are effective in thetreatment thereof. Such agents prolong the survival of the patient,inhibit the rapidly-proliferating cell growth associated with theneoplasm, or effect a regression of the neoplasm.

HDAC inhibitors are an emerging class of therapeutic agents that promotedifferentiation and apoptosis in hematologic and solid malignanciesthrough chromatin remodeling and gene expression regulation. SeveralHDAC inhibitors have been identified including benzamides (entinostat),short-chain fatty acids (i.e., Sodium phenylbutyrate); hydroxamic acids(i.e., suberoylanilide hydroxamic acid and thrichostatin A); cyclictetrapeptides containing a 2-amino-8-oxo-9,10-epoxy-decanoyl moiety(i.e., trapoxin A) and cyclic peptides without the 2-aminooxo-9,10-epoxy-decanoyl moiety (i.e., FK228). Entinostat is a benzamideHDAC inhibitor undergoing clinical investigation in multiple types ofsolid tumors and hematologic cancers. Entinostat is rapidly absorbed andhas a half-life of about 100 hours and, importantly, changes in histoneacetylation persist for several weeks following the administration ofentinostat.

Without being bound by any theory, it is contemplated that myeloidderived suppressor cells block effector anti-tumor T-cell activity andfacilitate immune evasion. Myeloid derived suppressor cells areincreased in breast cancer patients, with the highest levels ofcirculating myeloid derived suppressor cells being present in patientswith metastatic disease. In the setting of metastatic breast cancer,patients with higher than average levels of peripheral blood myeloidderived suppressor cells following palliative systemic therapy hadshorter overall survival. In the setting of adjuvant chemotherapy inbreast cancer, reduced levels of circulating myeloid derived suppressorcells has been shown to be correlated with increased clinical efficacy.Functional studies in mouse models of breast cancer have found thatdepletion or inactivation of myeloid derived suppressor cells reducestumor growth and progression through development of an anti-tumor immuneresponse.

Without being bound by any theory, it is contemplated that depletingmyeloid derived suppressor cells can act as a treatment for cancerthrough generation of an immune response against the tumor.

Myeloid-Derived Suppressor Cells

Myeloid derived suppressor cells are a heterogeneous population ofimmature myeloid cells which inhibit innate and adaptive immunity.Myeloid derived suppressor cells can inhibit innate and adaptiveimmunity through mechanisms including depletion of arginine, productionof reactive nitrogen and oxygen species, and secretion of inhibitorycytokines.

Myeloid derived suppressor cells commonly express the cell surfacemarkers CD33 and CD11b and have reduced expression of HLA-DR (HLA-DRlo/negative). Non-limiting examples of myeloid derived suppressor cellsinclude monocytic myeloid derived suppressor cells (M-MDSCs) andpolymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). M-MDSCsexpress the cell surface marker CD14, are HLA-DR lo/negative, and do notexpress the cell surface marker CD15 in humans. PMN-MDSCs do not expressthe cell surface marker CD14 and express the cell surface marker CD15 inhumans.

Myeloid derived suppressor cells cause inhibition of immune activatorcells such as T lymphocytes, natural killer (NK) cells, and dendriticcells (DCs). Conversely, myeloid derived suppressor cells can bestimulatory to immune suppressor cells such as T_(h)2 T lymphocytes, Tregulatory cells (T_(reg)) and tumor-associated macrophages (TAMs).Myeloid derived suppressor cells can also secrete cytokines, such asIL-6, that promote MDSC expansion. The expansion of myeloid derivedsuppressor cells can lead to sequestration of essential amino acids suchas arginine and cysteine necessary for the survival of T lymphocytes.Myeloid derived suppressor cells inhibit immunity through the productionof reactive oxygen species, such as nitric oxide, which are potentlytoxic to T lymphocytes.

Histone Deacetylases

The HDACs are a family including at least eighteen enzymes, grouped inthree classes (Class I, II and III). Class I HDACs include, but are notlimited to, HDACs 1, 2, 3, and 8. Class I HDACs can be found in thenucleus and are believed to be involved with transcriptional controlrepressors. Class II HDACs include, but are not limited to, HDACS 4, 5,6, 7, and 9 and can be found in both the cytoplasm as well as thenucleus. Class III HDACs are believed to be NAD dependent proteins andinclude, but are not limited to, members of the Sirtuin family ofproteins. Non-limiting examples of sirtuin proteins include SIRT1-7. Asused herein, the term “selective HDAC” refers to an HDAC inhibitor thatdoes not interact with all three HDAC classes.

HDAC Inhibitors

HDAC inhibitors can be classified broadly into pan HDAC inhibitors andselective HDAC inhibitors. Although there is a large structuraldiversity of known HDAC inhibitors, they share common features: a partthat interacts with the enzyme active site and a side-chain that sitsinside the channel leading to the active site. This can be seen with thehydroxamates such as SAHA, where the hydroxamate group is believed tointeract with the active site. In the case of the depsipeptides, it isbelieved that an intracellular reduction of the disulphide bond createsa free thiol group (which interacts with the active site) attached to a4-carbon alkenyl chain. A difference between the HDAC inhibitors is inthe way that they interact with the rim of the HDAC channel, which is atthe opposite end of the channel to the active site. It is thisinteraction, between the HDAC inhibitor and the rim of the channel,which is believed to account, at least in part, for some observeddifferences in HDAC selectivity between pan-HDAC inhibitors, such asSAHA and selective HDAC inhibitors such as the depsipeptides. Aparticularly preferred HDAC inhibitor is entinostat. Entinostat has thechemical nameN-(2-aminophenyl)-4-[N-(pyridine-3-yl)methoxycarbonylamino-methyl]-benzamideand the chemical structure shown below.

Programmed Cell Death-1 (PD-1)

PD-1 is a cell surface receptor that is a member of the CD28 family ofT-cell regulators, within the immunoglobulin superfamily of receptors.The human PD-1 gene is located at chromosome 2q37, and the full-lengthPD-1 cDNA encodes a protein with 288 amino acid residues with 60%homology to murine PD-1. It is present on CD4− CD8− (double negative)thymocytes during thymic development and is expressed upon activation inmature hematopoietic cells such as T and B cells, NKT cells andmonocytes after prolonged antigen exposure.

Without being bound by any theory, it is contemplated that binding ofthe ligand PD-L1 to PD-1 downregulates effector anti-tumor T-cellactivity and facilitates immune evasion. This is supported by thefinding of an association between PD-1/PD-L1 expression and poorprognosis in several tumor types including gastric, ovarian, lung andrenal carcinomas. PD-1 has been reported to be predominantly expressedby tumor infiltrating T lymphocytes, in melanoma.

In vitro studies of PD-1 blockade by PD-1-specific antibody showedaugmentation of cytotoxic T-cell responses to melanoma-specific antigensincluding increased frequencies of IFN-γ-secreting antigen-specificcells.

Without being bound by any theory, it is contemplated that targetingPD-1 may act as an effective therapeutic strategy for cancer.

The principal method for targeting PD-1 clinically has been through thedevelopment of genetically engineered monoclonal antibodies that inhibiteither PD-1 or PD-L1 function.

PD-L1 has also been shown to bind to B7-1 (CD80), an interaction thatalso suppresses T-cell proliferation and cytokine production; however,the exact relative contributions of the PD-L1: PD-1 and PD-L1: B7-1pathways in cancer remain unclear. The PD-1-targeting agents currentlyin development inhibit both pathways. However, as the binding sites forPD-1 and B7-1 are adjacent but not overlapping, agents that specificallytarget one or the other may potentially be developed.

Cancer cells drive high expression levels of PD-L1 on their surface,allowing activation of the inhibitory PD-1 receptor on any T cells thatinfiltrate the tumor microenvironment, effectively switching those cellsoff. Indeed, upregulation of PD-L1 expression levels has beendemonstrated in many different cancer types (eg, melanoma [40%-100%],NSCLC [35%-95%], and multiple myeloma [93%]), and high levels of PD-L1expression have been linked to poor clinical outcomes. Furthermore,tumor-infiltrating T cells have been shown to express significantlyhigher levels of PD-1 than T cells that infiltrate normal tissue. It isthought that the tumor microenvironment may secrete pro-inflammatorycytokines, including interferon-gamma (IFNγ) to upregulate theexpression of PD-1 on tumor-infiltrating T cells to ensure that they canrespond to the high levels of PD-L1 expressed on the tumor.

Pembrolizumab

Pembrolizumab is a humanized monoclonal IgG4 anti-PD-1 antibodyconsisting of a high-affinity mouse anti-PD-1-derived variable regiongrafted on to a human IgG4 immunoglobulin molecule with an engineered Fcregion for stabilization. Pre-clinical anti-tumor activity has beendemonstrated in animal models of multiple tumor types. A first-in-human,Phase I dose-escalation study was conducted in patients with advancedrefractory malignancies at dose levels 1, 3 and 10mg/kg givenintravenously initially and after 4 weeks and then every 2 weeks. Themaximum observed toxicity was grade 2 pruritus and no drug-related grade3 or greater adverse events (AEs) were observed. Therefore, the maximumtolerated dose was not reached. The half-life was 13.6-21.7 days and notobviously dose related. Four patients had some tumor regression. Thisstudy was then expanded, with patients receiving pembrolizumab at 10mg/kg every 2 weeks or either 2 or 10 mg/kg every 3 weeks innon-randomized cohorts; in total, there were 135 patients with melanoma.Enrollment included 48 patients who had received prior ipilimumab butcould not have experienced severe immune-related adverse events (irAEs).Though 79% of patients had some AEs, only 13% had severe (grade 3 or 4)drug-related toxicities including skin rash or pruritus, fatigue,diarrhea, abdominal pain and hepatic dysfunction. The highest rate ofsevere toxicities (23%) was in those receiving the highest dose (10mg/kg every 2 weeks) versus <10% in the less dose intense cohorts. AEspotentially of an autoimmune nature included isolated instances ofpneumonitis, kidney injury, hepatitis, diarrhea, hypothyroidism,hyperthyroidism and adrenal insufficiency. The overall objectiveresponse rate (ORR) based on immune-related response criteria was 38%(44 of 117) with 8 additional patients experiencing unconfirmedresponses. A total of 77% had some degree of tumor regression including8 patients with stable disease for over 24 weeks. The majority ofresponses were established by the time of the first radiologicassessment at 12 weeks. The median progression free survival exceeded 7months. Biopsies of responding tumors showed dense infiltration by CD8+T cells. Prior ipilimumab exposure did not appear to have an obviousimpact on efficacy or toxicity outcomes.

MPDL3280A

MPDL3280A is a human anti-PD-L1 mAb that contains an engineered fragmentcrystallizable (Fc) domain designed to optimize efficacy and safety byminimizing antibody-dependent cellular cytotoxicity (ADCC). Withoutbeing bound by any specific theory, it is understood that this structureallows inhibition of the PD-1/PD-L1 interaction, while minimizingADCC-mediated depletion of activated T cells that is required for aneffective antitumor immune response.

MPDL3280A has been evaluated in a phase I trial in patients with locallyadvanced or metastatic solid tumors. A total of 175 patients had beenrecruited to date. The antibody was administered as a single agent atescalating doses of ≤1, 3, 10, 15, and 20 mg/kg for a median duration of127 days. The results of two expansion cohorts have also been reported;a cohort of 85 patients (53 of whom were evaluable for efficacy) withsquamous or non-squamous NSCLC and a cohort of 45 metastatic melanomapatients (35 of whom were evaluable for efficacy). In both cohorts dosesof ≤1, 10, 15, and 25 mg/kg MPDL3280A were administered every 3 weeksfor up to 1 year. MPDL3280A demonstrated durable responses and was welltolerated; efficacy data are summarized in Table 1. Of the 85 patientsin the NSCLC cohort, 55% were heavily pretreated with at least threeprior therapies, and 81% were smokers or ex-smokers and 19% werenever-smokers. The 24-week PFS rate was 44% in squamous cell NSCLC and46% in non-squamous cell NSCLC.

Exemestane

Exemestane is a drug used to treat breast cancer. It is a member of theclass of drugs known as aromatase inhibitors. Some breast cancersrequire hormones to grow. Those cancers, known as hormonereceptor-positive breast cancers, express either estrogen receptors(ERs) or progesterone receptors (PRs), and thus termed as ER-positive orPR-positive breast cancers. The main source of estrogen inpre-menopausal women is the ovaries, while in post-menopausal women mostof the body's estrogen is produced via the conversion of androgens intoestrogen by the aromatase enzyme in peripheral tissues, such as inadipose mammary tissue and the brain. Exemestane is an aromataseinhibitor used in ER-positive breast cancer in addition to surgery orradiation in post-menopausal women. Exemestane is an irreversible, oralsteroidal aromatase inactivator, structurally related to the naturalsubstrate androstenedione. It acts as a false substrate for thearomatase enzyme, and is processed to an intermediate that bindsirreversibly to the aromatase active site to block aromatase by suicideinhibition.

Exemestane is indicated for the adjuvant treatment of post-menopausalwomen with ER-positive early breast cancer who have received two tothree years of tamoxifen. Subjects are switched to it for completion ofa total of five consecutive years of adjuvant hormonal therapy.Exemestane is also indicated for the treatment of advanced breast cancerin postmenopausal women whose disease has progressed following tamoxifentherapy. Oral exemestane at 25 mg per day for two to three years ofadjuvant therapy was generally more effective than five years ofcontinuous adjuvant tamoxifen in the treatment of post-menopausal womenwith early-stage ER-positive or unknown receptor status breast cancer.Exemestane at 25 mg per day is also effective in the primary adjuvantsetting of early-stage breast cancer in post-menopausal women.

Lung Cancer

Lung cancer is the leading cause of cancer deaths in women and men bothin the United States and throughout the world. Lung cancer has surpassedbreast cancer as the leading cause of cancer deaths in women. In theUnited States in 2014, 158,040 people were projected to die from lungcancer, which is more than the number of deaths from colon and rectal,breast, and prostate cancer combined. Only about 2% of those diagnosedwith lung cancer that has spread to other areas of the body are alivefive years after the diagnosis, although the survival rates for lungcancers diagnosed at the earliest stage are higher, with approximately49% surviving for five years or longer.

Cancer occurs when normal cells undergo a transformation that causesthem to grow and multiply without control. The cells form a mass ortumor that differs from the surrounding tissues from which it arises.Tumors are dangerous because they take oxygen, nutrients, and space fromhealthy cells and because they invade and destroy or reduce the abilityof normal tissues to function.

Most lung tumors are malignant. This means that they invade and destroythe healthy tissues around them and can spread throughout the body. Thetumors can spread to nearby lymph nodes or through the bloodstream toother organs. This process is called metastasis. When lung cancermetastasizes, the tumor in the lung is called the primary tumor, and thetumors in other parts of the body are called secondary tumors ormetastatic tumors.

Some tumors in the lung are metastatic from cancers elsewhere in thebody. The lungs are a common site for metastasis. If this is the case,the cancer is not considered to be lung cancer. For example, if prostatecancer spreads via the bloodstream to the lungs, it is metastaticprostate cancer (a secondary cancer) in the lung and is not called lungcancer.

Lung cancer comprises a group of different types of tumors. Lung cancersusually are divided into two main groups that account for about 95% ofall cases. The division into groups is based on the type of cells thatmake up the cancer. The two main types of lung cancer are characterizedby the cell size of the tumor when viewed under the microscope. They arecalled small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC). NSCLC includes several subtypes of tumors. SCLCs are lesscommon, but they grow more quickly and are more likely to metastasizethan NSCLCs. Often, SCLCs have already spread to other parts of the bodywhen the cancer is diagnosed. About 5% of lung cancers are of rare celltypes, including carcinoid tumor, lymphoma, and others. As used herein,the term “lung cancer” includes, but is not limited to, SCLC, NSCLC,carcinoid tumor, lymphoma, and their various subtypes.

Myeloid derived suppressor cells have several important functions inlung cancer. Pre-clinically, mouse models demonstrate that myeloidderived suppressor cells and lung tumors co-evolve in the course ofdisease inception and progression. In clinical NSCLC, numbers ofcirculating myeloid derived suppressor cells have been shown toinversely correlate with overall survival. M-MDSCs have been shown toproduce reactive oxygen species, inhibit T-cell proliferation, andsecrete interferon-γ (IFN-γ). Elevated levels of circulating myeloidderived suppressor cells and their activity inversely correlate withtherapeutic response and positively correlate with relapse in NSCLC.

Non-Small Cell Lung Cancer

NSCLC is a cancer of the lung which is not of the small cell carcinoma(oat cell carcinoma) type. The term “non-small cell lung cancer” appliesto the various types of bronchogenic carcinomas (those arising from thelining of the bronchi). Examples of specific types of NSCLC include, butare not limited to, adenocarcinoma, squamous cell carcinoma, and largecell cancer (i.e., large cell undifferentiated carcinoma).

Adenocarcinoma is a cancer that develops in the lining or inner surfaceof an organ. Adenocarcinoma is the most common type of lung cancer,making up 30%-40% of all cases of lung cancer. A subtype ofadenocarcinoma is called bronchoalveolar cell carcinoma, which creates apneumonia-like appearance on chest X-rays.

Squamous cell carcinoma is a cancer that begins in squamous cells.Squamos cells are thin, flat cells that look under the microscope likefish scales. Squamous cells are found in the tissue that forms thesurface of the skin, the lining of hollow organs of the body, and thepassages of the respiratory and digestive tracts. Squamous cellcarcinomas may arise in any of these tissues. Squamous cell carcinoma isthe second most common type of lung cancer, making up about 30% of allcases.

Large cell carcinoma shows no evidence of squamous or glandularmaturation. Thus these tumors are often diagnosed by default, when allother possibilities have been excluded. These tumors lack any diagnosticfeatures to suggest their diagnosis prior to biopsy. They tend to growrapidly, metastasize early, and are strongly associated with smoking.Large cell tumors are usually large, bulky, well-circumscribed,pink-grey masses with extensive hemorrhage and necrosis. Although theycommonly have central necrosis, they rarely cavitate. They tend topresent in the mid to peripheral lung zones. They may extend locally toinvolve the segmental or subsegmental bronchi. A variant of large cellcarcinoma is giant cell carcinoma. This subtype is particularlyaggressive and carries a very poor prognosis. These tumors generallypresent as a large peripheral mass with a focal necrotic component. Theydo not involve the large airways, unless by direct extension. Large cellcancer makes up 10%-20% of all cases of lung cancer.

Melanoma

Melanoma is a malignant tumor of melanocytes, which are the cells thatmake the pigment melanin and are derived from the neural crest. Althoughmost melanomas arise in the skin, they may also arise from mucosalsurfaces or at other sites to which neural crest cells migrate,including the uveal tract. Uveal melanomas differ significantly fromcutaneous melanoma in incidence, prognostic factors, molecularcharacteristics, and treatment.

In the United States in 2014, 9,710 people were projected to die frommelanoma, and numbers of new cases were estimated to be 76,100. Skincancer is the most common malignancy diagnosed in the United States,with 3.5 million cancers diagnosed in 2 million people annually.Melanoma represents less than 5% of skin cancers but results in mostdeaths. The incidence has been increasing over the past four decades.Elderly men are at highest risk; however, melanoma is the most commoncancer in young adults aged 25 to 29 years and the second most commoncancer in those aged 15 to 29 years. Ocular melanoma is the most commoncancer of the eye, with approximately 2,000 cases diagnosed annually.

Melanoma occurs predominantly in adults, and more than 50% of the casesarise in apparently normal areas of the skin. Although melanoma canoccur anywhere, including on mucosal surfaces and the uvea, melanoma inwomen occurs more commonly on the extremities, and in men it occurs mostcommonly on the trunk or head and neck.

Prognosis is affected by the characteristics of primary and metastatictumors. The most important prognostic factors include, but are notlimited to, the following: thickness or level of invasion of themelanoma, mitotic index, defined as mitoses per millimeter, ulcerationor bleeding at the primary site, number of regional lymph nodesinvolved, with distinction of macrometastasis and micrometastasis,systemic metastasis, site—nonvisceral versus lung versus all othervisceral sites, elevated serum lactate dehydrogenase level. Withoutbeing bound by any theory, it is contemplated that the presence of tumorinfiltrating lymphocytes can be a potential prognostic factor.

Myeloid derived suppressor cells have been shown to have severalimportant clinical correlations in melanoma. In the clinic, levels ofcirculating M-MDSCs and PMN-MDSCs positively correlate with diseaseburden in malignant melanoma. Patients with later-stage melanoma (stages3-4) have higher levels of circulating M-MDSCs. Circulating levels ofM-MDSCs are inversely correlated with overall survival in advancedmelanoma. Circulating levels of M-MDSCs are further inversely correlatedwith reduced levels of activated antigen-specific T lymphocytes inpatients with advanced melanoma. Decreased activation of M-MDSCs andPMN-MDSCs positively correlates with improved therapeutic response inmelanoma patients treated with ipilumimab. Ipilumimab treatment has alsobeen shown to reduce levels of circulating PMN-MDSCs.

Breast Cancer

Breast cancer is cancer that develops from breast tissue. Signs ofbreast cancer may include a lump in the breast, a change in breastshape, dimpling of the skin, fluid coming from the nipple, or a redscaly patch of skin. In those with distant spread of the disease, theremay be bone pain, swollen lymph nodes, dyspnea, or jaundice. Outcomesfor breast cancer vary depending on the cancer type, extent of disease,and subject age. Worldwide, breast cancer is the leading type of cancerin women, accounting for 25% of all cases. In 2012 it resulted in 1.68million cases and 522,000 deaths. It is more common in developedcountries and is more than 100 times more common in women than in men.Breast cancers are classified by several grading systems. Each of thesesystems can influence the prognosis and can affect treatment. Breastcancer is usually classified primarily by its histological appearance.Most breast cancers are derived from the epithelium lining the ducts orlobules, and these cancers are classified as ductal or lobularcarcinoma. Carcinoma in situ is growth of low grade cancerous orprecancerous cells within a particular tissue compartment such as themammary duct without invasion of the surrounding tissue. In contrast,invasive carcinoma does not confine itself to the initial tissuecompartment.

Breast cancer staging using the TNM system is based on the size of thetumor (T), whether or not the tumor has spread to the adjacent lymphnodes (N), and whether the tumor has metastasized (M) to a more distantpart of the body. Larger size, nodal spread, and metastasis have alarger stage number and a worse prognosis. The main stages are stage 0,stages 1-3, and stage 4. Stage 0 is a pre-cancerous or marker condition,either ductal carcinoma in situ (DCIS) or lobular carcinoma in situ(LCIS). Stages 1-3 are within the breast or regional lymph nodes. Stage4 is metastatic cancer that has a less favorable prognosis.

Breast cancer cells have receptors on their surface and in theircytoplasm and nucleus. Chemical messengers such as hormones bind toreceptors, and this causes changes in the cell. Breast cancer cells canor cannot have three important receptors: estrogen receptor (ER),progesterone receptor (PR), and HER2. This leads to a division of breastcancers into hormone receptor-positive breast cancers or ER-/PR-positivebreast cancers, HER2-positive breast cancers, and triple negative breastcancers, which are negative for ER, PR, and HER2.

Myeloid derived suppressor cells have been shown to have severalimportant clinical correlations in breast cancer. In pre-clinical modelsof breast cancer, myeloid derived suppressor cell levels positivelycorrelate with tumor size and inversely correlate with T cells. In theclinic, baseline levels of circulating myeloid derived suppressor cellscorrelate with disease burden, metastatic spread, and reduced survivalin metastatic breast cancer. Baseline levels of circulating myeloidderived suppressor cells have been shown to correlate with response toadjuvant chemotherapy in HER2-negative breast cancer, with increasinglevels indicating poorer response to chemotherapy.

Hormone Receptor-Positive Breast Cancer

Hormones, such as estrogen and progesterone, promote the growth ofcancers that are hormone receptor-positive. About two out of three ofbreast cancers are hormone receptor-positive, as they contain receptorsfor the hormones estrogen (ER-positive breast cancers) or progesterone(PR-positive breast cancers). As these breast cancers depend upon thehormones for growth, therapies have been designed to either lowerestrogen levels or stop estrogen activity breast cancer cells.

Non-limiting examples of therapies that stop estrogen activity includetamoxifen, toremifene, and fulvestrant. Tamoxifen blocks estrogenbinding to estrogen receptors in breast cancer cells. While tamoxifenacts like an anti-estrogen in breast cells, it functions like anestrogen in other tissues, like the uterus and the bones. Because itacts like estrogen in some tissues but like an anti-estrogen in others,it is called a selective estrogen receptor modulator (SERM). Toremifeneis another SERM that is approved to treat metastatic breast cancer.Fulvestrant is a drug that first blocks the estrogen receptor andtriggers its degradation. Fulvestrant is not a SERM, as it acts like ananti-estrogen throughout the body. Fulvestrant is used to treatmetastatic breast cancer after other hormone therapies, for example,tamoxifen, have stopped working.

Aromatase inhibitors (AIs) function to block estrogen production inpost-menopausal women. Aromatase inhibitors work by blocking aromatase,which converts androgens generated by adipose tissue and the brain.Non-limiting examples of aromatase inhibitors include letrozole,anastrozole, and exemestane.

Triple Negative Breast Cancer

Triple-negative breast cancer, characterized by tumors that do notexpress estrogen receptor (ER), progesterone receptor (PR), or HER-2genes, represents an important clinical challenge because these cancersdo not respond to endocrine therapy or other available targeted agents.The metastatic potential in triple-negative breast cancer is similar tothat of other breast cancer subtypes, but these tumors are associatedwith a shorter median time to relapse and death. One important goal istherefore the identification of prognostic factors and markers toreliably select high and low risk subsets of patients withtriple-negative disease for different treatment approaches of subtypeswith differential responsiveness to specific agents. However, a reliableprognostic marker has been elusive, and markers have been inconsistentlyuseful. For example, epidermal growth factor receptor (EGFR) has beenstudied, but there is still a lack of agreement on a standard assay orcutoff for EGFR expression levels with respect to prognosis. Similarly,because triple-negative status is sometimes used as a surrogate forbasal-like breast cancer, specific basal markers have been explored.Indeed, trials designed to accrue patients with basal-like breast cancerusing ER/PR and HER-2 negativity may provide only an approximation ofthe triple-negative population and are sometimes reanalyzed using morespecific indicators like CK 5/6, EGFR status, and others, again marredby discordances.

Chemotherapy remains the mainstay of treatment of triple-negative breastcancer, but important limitations still need to be overcome in the nextfew years if any significant clinical strides are to be made. Currenttreatment strategies for triple-negative disease include anthracyclines,taxanes, ixabepilone, platinum agents, and biologic agents. Morerecently, EGFR inhibition has been proposed as a therapeutic mechanismin triple-negative breast cancer, again with mixed results. Agents thattarget poly(ADP-ribose) polymerase and androgen receptors have also beenproposed in these patients or subsets of them, and ongoing trials shouldresult in definitive guidance with respect to the value of these agentsin triple-negative disease. Triple-negative breast cancer is clearly adistinct clinical subtype, from the perspective of both ER and HER-2expression, but further subclassification is needed. At present, thereis not a clear, proven effective single agent that targets a definingvulnerability in triple-negative breast cancer.

Various subtypes of triple negative breast cancer includes basal likeTNBC (Basal like 1 and 2 (BL-1, BL-2), Immunomodulatory (IM)) andmesenchymal stem like triple negative breast cancer (MSL), and luminalandrogen receptor (LAR) subtype.

PD-L1 is expressed on many cancers including renal cell carcinoma,pancreatic cancer, ovarian cancer, gastric cancer, esophageal cancer,and hepatocellular carcinoma. Research has identified the expression ofPD-L1 in 50% (22 out of 44 of tumors evaluated in a breast cancerstudy). In 15 (34%) it was restricted to the tumor epithelium, whereasin 18 (41%) it was identified in tumor infiltrating lymphocytes.Furthermore, it was found that intratumoral expression of PD-L1 wasassociated with high histologic grade and negative hormone receptorstatus. Consistent with the previous study, it was also in a separatestudy that approximately 20% of TNBC tumors express PD-L1. The majority(95%) of these TNBC tumors were grade 3.

Without being bound by any specific theory it is hypothesized that apossible mechanism by which tumors can drive PD-L1 expression is byoncogenic signaling pathways. This was first demonstrated inglioblastomas where it was observed that PTEN loss was associated withincreased PD-L1 expression, suggesting the involvement of the PI3Kpathway. Because PTEN loss is commonly seen in TNBC, a studyinvestigated the relationship between PTEN and PD-L1 expression. Inapproximately 50% of TNBC tumors included in the breast cancer tissuemicroarrays where there was >5% PD-L1 expression, a loss of PTENstaining was observed. Similarly, in a panel of TNBC cell lines, it wasfound that two exemplary cell lines with PTEN loss, MDA-MB-468 andBT-549, had high cell surface PD-L1 expression. Together, these datasuggested that there are likely multiple mechanisms of PD-L1 regulationin TNBC.

Methods of Selecting Patients for Combination Therapy

In certain embodiments, a method of the present disclosure comprisesmeasuring myeloid derived suppressor cells and peripheral bloodmononuclear cells to determine administration of a combination therapycomprising entinostat and a second therapeutic agent to a patientdiagnosed with a cancer. In some embodiments, the method furthercomprises selecting the patient for combination therapy if the ratio ofmyeloid derived suppressor to peripheral blood mononuclear cells isbetween 1:200 and 1:4.

In certain embodiments, a method of the present disclosure comprisesmeasuring CD14-positive and HLA-DR-(lo/negative) cells, measuringCD14-positive cells, or measuring peripheral blood mononuclear cells todetermine administration of a combination therapy comprising entinostatand a second therapeutic agent to a patient diagnosed with a cancer. Insome embodiments, the method further comprises selecting the patient forcombination therapy if the ratio of CD14-positive andHLA-DR-(lo/negative) cells to peripheral blood mononuclear cells isbetween 1:200 and 1:1. In some embodiments, the method further comprisesselecting the patient for combination therapy if the ratio ofCD14-positive and HLA-DR-(lo/negative) cells to CD14-positive cells isbetween 1:100 and 99:1.

Non-limiting examples of the second therapeutic agent include anti-PD-1antibodies, for example, nivolumab and pembrolizumab; anti-PD-L1antibodies, such as MPDL3280A; and exemestane. Non-limiting examples ofthe cancer include breast cancers, for example, hormonereceptor-positive breast cancers and triple negative breast cancers;lung cancers, for example, non-small cell lung cancers, squamous cellcarcinomas, and large cell carcinomas; and melanomas, for example,metastatic melanomas. In some embodiments, the second therapeutic agentis MPDL3280A and the cancer is a breast cancer. In some embodiments, thesecond therapeutic agent is MPDL3280A and the breast cancer is atriple-negative breast cancer. In some embodiments, the secondtherapeutic agent is exemestane and the cancer is a breast cancer. Insome embodiments, the second therapeutic agent is exemestane and thebreast cancer is a hormone receptor-positive breast cancer.

In some embodiments, the entinostat and exemestane are administeredorally. In some embodiments, the entinostat is administered orally andthe anti-PD-1 antibody or anti-PD-L1 antibody is administered by aninfusion. Non-limiting examples of infusions include subcutaneousinfusion, intravenous infusion, intraperitoneal infusion, and infusionby osmotic pump.

In some embodiments, the entinostat is administered first in thecombination therapy. In some embodiments, the entinostat is administeredweekly. In some embodiments, the entinostat is administered every twoweeks.

Entinostat, exemestane, anti-PD-1 antibody, or anti-PD-L1 antibody canbe administered about every day, about every two days, about every threedays, about every four days, about every five days, about every sixdays, about every week, about every two weeks, about every three weeks,about every four weeks, about every month, about every five weeks, aboutevery six weeks, about every seven weeks, about every eight weeks, orabout every two months. Entinostat, exemestane, anti-PD-1 antibody, oranti-PD-L1 antibody can be administered from about every day to aboutevery two days, from about every two days to about every three days,from about every three days to about every four days, from about everyfour days to about every five days, from about every five days to aboutevery six days, from about every six days to about every week, fromabout every week to about every two weeks, from about every two weeks toabout every three weeks, from about every three weeks to about everyfour weeks, from about every four weeks to about every month, from aboutevery month to about every five weeks, from about every five weeks toabout every six weeks, from about every six weeks to about every sevenweeks, from about every seven weeks to about every eight weeks, or fromabout every eight weeks to about every two months.

In some embodiments, the myeloid derived suppressor cells and peripheralblood mononuclear cells are circulating and are each measured inperipheral blood by obtaining a peripheral blood sample. In someembodiments, the CD14-positive and HLA-DR-(lo/negative) cells,CD14-positive cells, and peripheral blood mononuclear cells arecirculating and are each measured in peripheral blood by obtaining aperipheral blood sample. In some embodiments, the peripheral bloodsample is treated with an anticoagulant. In some embodiments, theperipheral blood sample is collected in or transferred into ananticoagulant-containing container. Non-limiting examples ofanticoagulants include heparin, sodium heparin, potassium oxalate, EDTA,and sodium citrate. In some embodiments, the peripheral blood sample istreated with a red blood cell lysis agent. In some embodiments, themyeloid derived suppressor cells and peripheral blood mononuclear cellsare measured in tissue biopsies.

In some embodiments, a number of myeloid derived suppressor cells andperipheral blood mononuclear cells are measured in the peripheral bloodsample and a percentage of myeloid derived suppressor cells relative toperipheral blood mononuclear cells is determined. In some embodiments,one or more myeloid derived suppressor cells from the peripheral bloodsample is contacted with a first binding agent to generate one or morefirst binding agent-myeloid derived suppressor cell complexes. In someembodiments, one or more peripheral blood mononuclear cells from theperipheral blood sample is contacted with a second binding agent togenerate one or more second binding agent-peripheral blood mononuclearcell complexes. In some embodiments, a percentage of the first bindingagent-myeloid derived suppressor cell complexes relative to the secondbinding agent-peripheral blood mononuclear cell complexes in theperipheral blood sample is measured.

In some embodiments, the percentage of myeloid derived suppressor cellsrelative to peripheral blood mononuclear cells, or that of first bindingagent-myeloid derived suppressor cell complexes and the second bindingagent-peripheral blood mononuclear cell complexes, in a peripheral bloodsample or a tissue biopsy is utilized to select patients foradministering a combination therapy comprising entinostat and a secondtherapeutic agent.

In some embodiments, the percentage of myeloid derived suppressor orperipheral blood mononuclear cells, or that of first bindingagent-myeloid derived suppressor cell complexes and the second bindingagent-peripheral blood mononuclear cell complexes, in a peripheral bloodsample or a tissue biopsy is at least about 0.1%, at least about 0.2%,at least about 0.3%, at least about 0.4%, at least about 0.5%, at leastabout 0.6%, at least about 0.7%, at least about 0.8%, at least about0.9%, at least about 1%, at least about 1.1%, at least about 1.2%, atleast about 1.3%, at least about 1.4%, at least about 1.5%, at leastabout 1.6%, at least about 1.7%, at least about 1.8%, at least about1.9%, at least about 2%, at least about 3%, at least about 4%, at leastabout 5%, at least about 6%, at least about 7%, at least about 8%, atleast about 9%, at least about 10%, at least about 11%, at least about12%, at least about 13%, at least about 14%, at least about 15%, atleast about 16%, at least about 17%, at least about 18%, at least about19%, at least about 20%, at least about 21%, at least about 22%, atleast about 23%, at least about 24%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, or atleast about 50%.

In some embodiments, the percentage of myeloid derived suppressor cellsrelative to peripheral blood mononuclear cells, or that of first bindingagent-myeloid derived suppressor cell complexes and the second bindingagent-peripheral blood mononuclear cell complexes, in a peripheral bloodsample or a tissue biopsy myeloid derived suppressor cells is from about0.1% to about 0.2%, from about 0.2% to about 0.3%, from about 0.3% toabout 0.4%, from about 0.4% to about 0.5%, from about 0.5% to about0.6%, from about 0.6% to about 0.7%, from about 0.7% to about 0.8%, fromabout 0.8% to about 0.9%, from about 0.9% to about 1%, from about 1% toabout 1.1%, from about 1.1% to about 1.2%, from about 1.2% to about1.3%, from about 1.4% to about 1.5%, from about 1.5% to about 1.6%, fromabout 1.6% to about 1.7%, from about 1.7% to about 1.8%, from about 1.8%to about 1.9%, from about 1.9% to about 2%, from about 2% to about 3%,from about 3% to about 4%, from about 4% to about 5%, from about 5% toabout 6%, from about 6% to about 7%, from about 7% to about 8%, fromabout 8% to about 9%, from about 9% to about 10%, from about 10% toabout 11%, from about 11% to about 12%, from about 12% to about 13%,from about 13% to about 14%, from about 14% to about 15%, from about 15%to about 16%, from about 16% to about 17%, from about 17% to about 18%,from about 18% to about 19%, from about 19% to about 20%, from about 20%to about 21%, from about 21% to about 22%, from about 22% to about 23%,from about 23% to about 24%, from about 24% to about 25%, from about 25%to about 30%, from about 30% to about 35%, from about 35% to about 40%,from about 40% to about 45%, or from about 45% to about 50%.

In some embodiments, a number of myeloid derived suppressor cells andperipheral blood mononuclear cells are measured in the peripheral bloodsample and a ratio of myeloid derived suppressor cells to peripheralblood mononuclear cells is determined. In some embodiments, one or moremyeloid derived suppressor cells from the peripheral blood sample iscontacted with a first binding agent to generate one or more firstbinding agent-myeloid derived suppressor cell complexes. In someembodiments, one or more peripheral blood mononuclear cells from theperipheral blood sample is contacted with a second binding agent togenerate one or more second binding agent-peripheral blood mononuclearcell complexes. In some embodiments, a ratio of the first bindingagent-myeloid derived suppressor cell complexes and the second bindingagent-peripheral blood mononuclear cell complexes in the peripheralblood sample is measured. In some embodiments, the ratio of myeloidderived suppressor cells to peripheral blood mononuclear cells, or thatof first binding agent-myeloid derived suppressor cell complexes and thesecond binding agent-peripheral blood mononuclear cell complexes, isutilized to select patients for administering a combination therapycomprising entinostat and a second therapeutic agent.

In some embodiments, the ratio of first binding agent-myeloid derivedsuppressor cell complexes and the second binding agent-peripheral bloodmononuclear cell complexes utilized to select patients for administeringa combination therapy comprising entinostat and a second therapeuticagent.

In some embodiments, the ratio of myeloid derived suppressor cellsrelative to peripheral blood mononuclear cells, or that of first bindingagent-myeloid derived suppressor cell complexes and the second bindingagent-peripheral blood mononuclear cell complexes in a peripheral bloodsample or a tissue biopsy is 1:10000, 1:5000, 1:4000, 1:3000, 1:2000,1:1000, 1:500, 1:400, 1:300, 1:250, 1:200, 1:150, 1:100, 1:90, 1:80,1:70, 1:60, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41,1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29,1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17,1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3, 1:2, or 1:1.

In some embodiments, the ratio of myeloid derived suppressor cellsrelative to peripheral blood mononuclear cells, or that of first bindingagent-myeloid derived suppressor cell complexes and the second bindingagent-peripheral blood mononuclear cell complexes in a peripheral bloodsample or a tissue biopsy is from 1:10000 to 1:5000, from 1:5000 to1:4000, from 1:4000 to 1:3000, from 1:3000 to 1:2000, from 1:2000 to1:1000, from 1:1000 to 1:500, from 1:500 to 1:400, from 1:400 to 1:300,from 1:300 to 1:200, from 1:200 to 1:100, from 1:100 to 1:90, from 1:90to 1:80, from 1:80 to 1:70, from 1:70 to 1:60, from 1:60 to 1:50, from1:50 to 1:49, from 1:49 to 1:48, from 1:48 to 1:47, from 1:47 to 1:46,from 1:46 to 1:45, from 1:45 to 1:44, from 1:44 to 1:43, from 1:43 to1:42, from 1:42 to 1:41, from 1:41 to 1:40, from 1:40 to 1:39, from 1:39to 1:38, from 1:38 to 1:37, from 1:37 to 1:36, from 1:36 to 1:35, from1:35 to 1:34, from 1:34 to 1:33, from 1:33 to 1:32, from 1:32 to 1:31,from 1:31 to 1:30, from 1:30 to 1:29, from 1:29 to 1:28, from 1:28 to1:27, from 1:27 to 1:26, from 1:26 to 1:25, from 1:25 to 1:24, from 1:24to 1:23, from 1:23 to 1:22, from 1:22 to 1:21, from 1:21 to 1:20, from1:20 to 1:19, from 1:19 to 1:18, from 1:18 to 1:17, from 1:17 to 1:16,from 1:16 to 1:15, from 1:15 to 1:14, from 1:14 to 1:13, from 1:13 to1:12, from 1:12 to 1:11, from 1:11 to 1:10, from 1:10 to 1:9, from 1:9to 1:8, from 1:8 to 1:7, from 1:7 to 1:6, from 1:6 to 1:5, from 1:5 to1:4, from 1:4 to 1:3, from 1:3 to 1:2, or from 1:2 to 1:1.

In some embodiments, a number of myeloid derived suppressor cells perunit volume of a biological sample is determined. Non-limiting examplesof unit volumes include picoliters (pL), nanoliters (nL), microliters(μL), milliliters (mL), deciliters (dL), and liters (L). In someembodiments, a number of myeloid derived suppressor cells from theperipheral blood sample is contacted with a binding agent to generate anumber of binding agent-myeloid derived suppressor cell complexes perunit volume. In some embodiments, the number of myeloid derivedsuppressor cells or binding agent-myeloid derived suppressor cellcomplexes per unit volume of the biological sample utilized to selectpatients for administering a combination therapy comprising entinostatand a second therapeutic agent. In some embodiments, the biologicalsample is peripheral blood sample.

In some embodiments, the myeloid derived suppressor cells or bindingagent-myeloid derived suppressor cell complexes per unit volume of thebiological sample is about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 15, about 20, about 25,about 30, about 35, about 40, about 45, about 50, about 60, about 70,about 80, about 90, about 100, about 150, about 200, about 250, about300, about 350, about 400, about 450, about 500, about 600, about 700,about 800, about 900, about 1000, about 1500, about 2000, about 2500,about 3000, about 3500, about 4000, about 4500, about 5000, about 6000,about 7000, about 8000, about 9000, about 10000, about 15000, about20000, about 25000, about 30000, about 35000, about 40000, about 45000,about 50000, about 60000, about 70000, about 80000, about 90000, orabout 100000 per unit volume.

In some embodiments, the myeloid derived suppressor cells or bindingagent-myeloid derived suppressor cell complexes per unit volume of thebiological sample is from about 1 to about 2, from about 2 to about 3,from about 3 to about 4, from about 4 to about 5, from about 5 to about6, from about 6 to about 7, from about 7 to about 8, from about 8 toabout 9, from about 9 to about 10, from about 10 to about 15, from about15 to about 20, from about 20 to about 25, from about 25 to about 30,from about 30 to about 35, from about 35 to about 40, from about 40 toabout 45, from about 45 to about 50, from about 50 to about 60, fromabout 60 to about 70, from about 70 to about 80, from about 80 to about90, from about 90 to about 100, from about 100 to about 150, from about150 to about 200, from about 200 to about 250, from about 250 to about300, from about 300 to about 350, from about 350 to about 400, fromabout 400 to about 450, from about 450 to about 500, from about 500 toabout 600, from about 600 to about 700, from about 700 to about 800,from about 800 to about 900, from about 900 to about 1000, from about1000 to about 1500, from about 1500 to about 2000, from about 2000 toabout 2500, from about 2500 to about 3000, from about 3000 to about3500, from about 3500 to about 4000, from about 4000 to about 4500, fromabout 4500 to about 5000, from about 5000 to about 6000, from about 6000to about 7000, from about 7000 to about 8000, from about 8000 to about9000, from about 9000 to about 10000, from about 10000 to about 15000,from about 15000 to about 20000, from about 20000 to about 25000, fromabout 25000 to about 30000, from about 30000 to about 35000, from about35000 to about 40000, from about 40000 to about 45000, from about 45000to about 50000, from about 50000 to about 60000, from about 60000 toabout 70000, from about 70000 to about 80000, from about 80000 to about90000, or from about 90000 to about 100000 per unit volume.

In some embodiments, a number of CD14-positive and HLA-DR-(lo/negative)cells, CD14-positive cells, and peripheral blood mononuclear cells aremeasured in the peripheral blood sample and a percentage ofCD14-positive and HLA-DR-(lo/negative) cells, CD14-positive cells, andperipheral blood mononuclear cells is determined.

In some embodiments, the percentage of CD14-positive andHLA-DR-(lo/negative) cells relative to either CD14-positive cells orperipheral blood mononuclear cells is utilized to select patients foradministering a combination therapy comprising entinostat and a secondtherapeutic agent.

In some embodiments, the percentage of CD14-positive andHLA-DR-(lo/negative) cells relative to either CD14-positive cells orperipheral blood mononuclear cells in a peripheral blood sample or atissue biopsy is at least about 0.1%, at least about 0.2%, at leastabout 0.3%, at least about 0.4%, at least about 0.5%, at least about0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, atleast about 1%, at least about 1.1%, at least about 1.2%, at least about1.3%, at least about 1.4%, at least about 1.5%, at least about 1.6%, atleast about 1.7%, at least about 1.8%, at least about 1.9%, at leastabout 2%, at least about 3%, at least about 4%, at least about 5%, atleast about 6%, at least about 7%, at least about 8%, at least about 9%,at least about 10%, at least about 11%, at least about 12%, at leastabout 13%, at least about 14%, at least about 15%, at least about 16%,at least about 17%, at least about 18%, at least about 19%, at leastabout 20%, at least about 21%, at least about 22%, at least about 23%,at least about 24%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%.

In some embodiments, the percentage of CD14-positive andHLA-DR-(lo/negative) cells relative to either CD14-positive cells orperipheral blood mononuclear cells in a peripheral blood sample or atissue biopsy is from about 0.1% to about 0.2%, from about 0.2% to about0.3%, from about 0.3% to about 0.4%, from about 0.4% to about 0.5%, fromabout 0.5% to about 0.6%, from about 0.6% to about 0.7%, from about 0.7%to about 0.8%, from about 0.8% to about 0.9%, from about 0.9% to about1%, from about 1% to about 1.1%, from about 1.1% to about 1.2%, fromabout 1.2% to about 1.3%, from about 1.4% to about 1.5%, from about 1.5%to about 1.6%, from about 1.6% to about 1.7%, from about 1.7% to about1.8%, from about 1.8% to about 1.9%, from about 1.9% to about 2%, fromabout 2% to about 3%, from about 3% to about 4%, from about 4% to about5%, from about 5% to about 6%, from about 6% to about 7%, from about 7%to about 8%, from about 8% to about 9%, from about 9% to about 10%, fromabout 10% to about 11%, from about 11% to about 12%, from about 12% toabout 13%, from about 13% to about 14%, from about 14% to about 15%,from about 15% to about 16%, from about 16% to about 17%, from about 17%to about 18%, from about 18% to about 19%, from about 19% to about 20%,from about 20% to about 21%, from about 21% to about 22%, from about 22%to about 23%, from about 23% to about 24%, from about 24% to about 25%,from about 25% to about 30%, from about 30% to about 35%, from about 35%to about 40%, from about 40% to about 45%, from about 45% to about 50%,from about 50% to about 60%, from about 60% to about 70%, from about 70%to about 80%, from about 80% to about 90%, from about 90% to about 95%,from about 95% to about 96%, from about 96% to about 97%, from about 97%to about 98%, or from about 98% to about 99%.

In some embodiments, a number of CD14-positive and HLA-DR-(lo/negative)cells, CD14-positive cells, and peripheral blood mononuclear cells aremeasured in the peripheral blood sample and a ratio of CD14-positive andHLA-DR-(lo/negative) cells relative to either CD14-positive cells orperipheral blood mononuclear cells is determined. In some embodiments,the ratio of CD14-positive and HLA-DR-(lo/negative) cells relative toeither CD14-positive cells or peripheral blood mononuclear cells isutilized to select patients for administering a combination therapycomprising entinostat and a second therapeutic agent.

In some embodiments, the ratio of CD14-positive and HLA-DR-(lo/negative)cells relative to either CD14-positive cells or peripheral bloodmononuclear cells in a peripheral blood sample or a tissue biopsy is1:10000, 1:5000, 1:4000, 1:3000, 1:2000, 1:1000, 1:500, 1:400, 1:300,1:250, 1:200, 1:150, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:49, 1:48,1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36,1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24,1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12,1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1,9:1, 19:1, 24:1, 97:1, 49:1, or 99:1.

In some embodiments, the ratio of CD14-positive and HLA-DR-(lo/negative)cells relative to either CD14-positive cells or peripheral bloodmononuclear cells in a peripheral blood sample or a tissue biopsy isfrom 1:10000 to 1:5000, from 1:5000 to 1:4000, from 1:4000 to 1:3000,from 1:3000 to 1:2000, from 1:2000 to 1:1000, from 1:1000 to 1:500, from1:500 to 1:400, from 1:400 to 1:300, from 1:300 to 1:200, from 1:200 to1:100, from 1:100 to 1:90, from 1:90 to 1:80, from 1:80 to 1:70, from1:70 to 1:60, from 1:60 to 1:50, from 1:50 to 1:49, from 1:49 to 1:48,from 1:48 to 1:47, from 1:47 to 1:46, from 1:46 to 1:45, from 1:45 to1:44, from 1:44 to 1:43, from 1:43 to 1:42, from 1:42 to 1:41, from 1:41to 1:40, from 1:40 to 1:39, from 1:39 to 1:38, from 1:38 to 1:37, from1:37 to 1:36, from 1:36 to 1:35, from 1:35 to 1:34, from 1:34 to 1:33,from 1:33 to 1:32, from 1:32 to 1:31, from 1:31 to 1:30, from 1:30 to1:29, from 1:29 to 1:28, from 1:28 to 1:27, from 1:27 to 1:26, from 1:26to 1:25, from 1:25 to 1:24, from 1:24 to 1:23, from 1:23 to 1:22, from1:22 to 1:21, from 1:21 to 1:20, from 1:20 to 1:19, from 1:19 to 1:18,from 1:18 to 1:17, from 1:17 to 1:16, from 1:16 to 1:15, from 1:15 to1:14, from 1:14 to 1:13, from 1:13 to 1:12, from 1:12 to 1:11, from 1:11to 1:10, from 1:10 to 1:9, from 1:9 to 1:8, from 1:8 to 1:7, from 1:7 to1:6, from 1:6 to 1:5, from 1:5 to 1:4, from 1:4 to 1:3, from 1:3 to 1:2,from 1:2 to 1:1, from 1:1 to 2:1, from 2:1 to 3:1, from 3:1 to 4:1, from4:1 to 9:1, from 9:1 to 19:1, from 19:1 to 24:1, from 24:1 to 97:1, from97:1 to 49:1, or from 49:1 to 99:1.

In some embodiments, a number of CD14-positive and HLA-DR-(lo/negative)cells per unit volume of a biological sample is determined. Non-limitingexamples of unit volumes include picoliters (pL), nanoliters (nL),microliters (4), milliliters (mL), deciliters (dL), and liters (L). Insome embodiments, the number of CD14-positive and HLA-DR-(lo/negative)cells per unit volume of the biological sample is utilized to selectpatients for administering a combination therapy comprising entinostatand a second therapeutic agent. In some embodiments, the biologicalsample is peripheral blood sample.

In some embodiments, the number of CD14-positive andHLA-DR-(lo/negative) cells per unit volume of the biological sample isabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 15, about 20, about 25, about 30, about 35,about 40, about 45, about 50, about 60, about 70, about 80, about 90,about 100, about 150, about 200, about 250, about 300, about 350, about400, about 450, about 500, about 600, about 700, about 800, about 900,about 1000, about 1500, about 2000, about 2500, about 3000, about 3500,about 4000, about 4500, about 5000, about 6000, about 7000, about 8000,about 9000, about 10000, about 15000, about 20000, about 25000, about30000, about 35000, about 40000, about 45000, about 50000, about 60000,about 70000, about 80000, about 90000, or about 100000 per unit volume.

In some embodiments, the number of CD14-positive andHLA-DR-(lo/negative) cells per unit volume of the biological sample isfrom about 1 to about 2, from about 2 to about 3, from about 3 to about4, from about 4 to about 5, from about 5 to about 6, from about 6 toabout 7, from about 7 to about 8, from about 8 to about 9, from about 9to about 10, from about 10 to about 15, from about 15 to about 20, fromabout 20 to about 25, from about 25 to about 30, from about 30 to about35, from about 35 to about 40, from about 40 to about 45, from about 45to about 50, from about 50 to about 60, from about 60 to about 70, fromabout 70 to about 80, from about 80 to about 90, from about 90 to about100, from about 100 to about 150, from about 150 to about 200, fromabout 200 to about 250, from about 250 to about 300, from about 300 toabout 350, from about 350 to about 400, from about 400 to about 450,from about 450 to about 500, from about 500 to about 600, from about 600to about 700, from about 700 to about 800, from about 800 to about 900,from about 900 to about 1000, from about 1000 to about 1500, from about1500 to about 2000, from about 2000 to about 2500, from about 2500 toabout 3000, from about 3000 to about 3500, from about 3500 to about4000, from about 4000 to about 4500, from about 4500 to about 5000, fromabout 5000 to about 6000, from about 6000 to about 7000, from about 7000to about 8000, from about 8000 to about 9000, from about 9000 to about10000, from about 10000 to about 15000, from about 15000 to about 20000,from about 20000 to about 25000, from about 25000 to about 30000, fromabout 30000 to about 35000, from about 35000 to about 40000, from about40000 to about 45000, from about 45000 to about 50000, from about 50000to about 60000, from about 60000 to about 70000, from about 70000 toabout 80000, from about 80000 to about 90000, or from about 90000 toabout 100000 per unit volume.

In some embodiments, myeloid derived suppressor cells, CD14-positive andHLA-DR-(lo/negative) cells, CD14-positive cells, and/or peripheral bloodmononuclear cells are measured using flow cytometry, mass cytometry,cytospin, or immunohistochemistry.

Flow cytometry is a laser-based technology used in cell counting, cellsorting, and biomarker detection, by suspending cells in a stream offluid and passing them by an electronic detection apparatus. It allowssimultaneous multiparametric analysis of the physical and chemicalcharacteristics of up to thousands of particles per second.

Mass cytometry is a mass spectrometry technique based on inductivelycoupled plasma mass spectrometry for the determination cell identity andfunction. In this technology, binding agents are tagged withisotopically pure rare earth elements. These binding agents are thenapplied to tag cells and their components. Cells are nebulized and sentthrough an argon plasma laser, ionizing the multi-atom rare earthelemental tags. The ionized, tagged cells are then analyzed by atime-of-flight mass spectrometer. The advantage of mass cytometry is thecapacity to overcome the limitations developed by spectral overlap inflow cytometry.

Cytospin is a technique in which suspension cells are centrifuged ontoglass slides as a smear for cell staining and cell counting.Concentrated cell suspensions that exist in a low-viscosity medium makegood candidates for smear preparations. Dilute cell suspensions existingin a dilute medium are best suited for the preparation of cytospinsthrough cytocentrifugation. Cell suspensions that exist in ahigh-viscosity medium, are best suited to be tested as swabpreparations. The constant among these preparations is that the wholecell is present on the slide surface. Immunohistochemistry is a type ofhistological staining for detecting antigens in cells of a tissuesection by exploiting the principle of antibodies binding specificallyto antigens in biological tissues. Visualizing an antibody-antigeninteraction can be accomplished in a number of ways. The antibody can beconjugated to an enzyme, such as peroxidase, that can catalyze acolor-producing reaction. Alternatively, the antibody can be tagged to afluorophore, such as fluorescein or rhodamine.

In some embodiments, flow cytometry, mass cytometry, cytospin, orimmunohistochemistry is used to detect cells such as myeloid derivedsuppressor cells or peripheral blood mononuclear cells. In someembodiments, flow cytometry, mass cytometry, cytospin, orimmunohistochemistry include use of a binding agent to create a bindingagent-myeloid derived suppressor cells and a binding agent-peripheralblood mononuclear cells complex. In some embodiments, the binding agentis used to identify myeloid derived suppressor cells and peripheralblood mononuclear cells by a cell surface marker. In some embodiments,the binding agent is an antibody.

In some embodiments, the number of binding agents bound to a myeloidderived suppressor cell or peripheral blood mononuclear cell is at leastabout 1, at least about 2, at least about 3, at least about 4, at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 16,at least about 20, least about 30, least about 40, at least about 50, atleast about 60, at least about 70, least about 80, least about 90, atleast about 100, at least about 125, at least about 150, at least about175, or at least about 200. In some embodiments, the number of bindingagents bound to a myeloid derived suppressor cell or peripheral bloodmononuclear cell is from about 1 to about 2, from about 2 to about 3,from about 3 to about 4, from about 4 to about 5, from about 5 to about6, from about 6 to about 7, from about 7 to about 8, from about 8 toabout 9, from about 9 to about 10, from about 10 to about 11, from about11 to about 12, from about 12 to about 13, from about 14 to about 15,from about 15 to about 16, from about 16 to about 20, from about 20 toabout 30, from about 30 to about 40, from about 40 to about 50, fromabout 50 to about 60, from about 60 to about 70, from about 70 to about80, from about 80 to about 90, from about 90 to about 100, from about100 to about 125, from about 125 to about 150, from about 150 to about175, or from about 175 to about 200.

In some embodiments, myeloid derived suppressor cells and peripheralblood mononuclear cells, M-MDSCs, or PMN-MDSCs are identified by a cellsurface marker. Non-limiting examples of cell surface markers thatidentify myeloid derived suppressor cells include CD11b, CD33, and CD40.Non-limiting examples of cell surface markers that identify peripheralblood mononuclear cells include CD3, CD14, CD19, CD56, and HLA-DR.M-MDSCs can be identified by any combination of the myeloid derivedsuppressor cell markers combined with CD14. PMN-MDSCs can be identifiedby any combination of the myeloid derived suppressor cell markerscombined with CD15. In some embodiments, the myeloid derived suppressorcells, peripheral blood mononuclear cells, M-MDSCs, or PMN-MDSCs arecontacted by a binding agent to form a cell-binding agent complex. Thebinding agent can bind any of the foregoing cell surface markers or anycombination thereof.

In some embodiments, myeloid derived suppressor cells are identified byCD11b and peripheral blood mononuclear cells are identified by CD3. Insome embodiments, myeloid derived suppressor cells are identified byCD11b and Peripheral blood mononuclear cells are identified by CD14. Insome embodiments, myeloid derived suppressor cells are identified byCD11b and peripheral blood mononuclear cells are identified by CD19. Insome embodiments, myeloid derived suppressor cells are identified byCD11b and peripheral blood mononuclear cells are identified by CD56. Insome embodiments, myeloid derived suppressor cells are identified byCD11b and peripheral blood mononuclear cells are identified by HLA-DR.In some embodiments, myeloid derived suppressor cells are identified byCD33 and peripheral blood mononuclear cells are identified by CD3. Insome embodiments, myeloid derived suppressor cells are identified byCD33 and peripheral blood mononuclear cells are identified by CD14. Insome embodiments, myeloid derived suppressor cells are identified byCD33 and peripheral blood mononuclear cells are identified by CD19. Insome embodiments, myeloid derived suppressor cells are identified byCD33 and peripheral blood mononuclear cells are identified by CD56. Insome embodiments, myeloid derived suppressor cells are identified byCD33 and peripheral blood mononuclear cells are identified by HLA-DR. Insome embodiments, myeloid derived suppressor cells are identified byCD40 and peripheral blood mononuclear cells are identified by CD3. Insome embodiments, myeloid derived suppressor cells are identified byCD40 and peripheral blood mononuclear cells are identified by CD14. Insome embodiments, myeloid derived suppressor cells are identified byCD40 and peripheral blood mononuclear cells are identified by CD19. Insome embodiments, myeloid derived suppressor cells are identified byCD40 and peripheral blood mononuclear cells are identified by CD56. Insome embodiments, myeloid derived suppressor cells are identified byCD40 and peripheral blood mononuclear cells are identified by HLA-DR. Insome embodiments, myeloid derived suppressor cells are identified byCD11b and CD33 and peripheral blood mononuclear cells are identified byCD3. In some embodiments, myeloid derived suppressor cells areidentified by CD11b and CD33 and peripheral blood mononuclear cells areidentified by CD14. In some embodiments, myeloid derived suppressorcells are identified by CD11b and CD33 and peripheral blood mononuclearcells are identified by CD19. In some embodiments, myeloid derivedsuppressor cells are identified by CD11b and CD33 and peripheral bloodmononuclear cells are identified by CD56. In some embodiments, myeloidderived suppressor cells are identified by CD11b and CD33 and peripheralblood mononuclear cells are identified by HLA-DR. In some embodiments,myeloid derived suppressor cells are identified by CD11b and CD33 andperipheral blood mononuclear cells are identified by CD3, CD14, CD19,CD56, and HLA-DR.

In some embodiments, M-MDSCs are identified by CD11b and CD14 andperipheral blood mononuclear cells are identified by CD3. In someembodiments, M-MDSCs are identified by CD11b and CD14 and peripheralblood mononuclear cells are identified by CD14. In some embodiments,M-MDSCs are identified by CD11b and CD14 and peripheral bloodmononuclear cells are identified by CD19. In some embodiments, M-MDSCsare identified by CD11b and CD14 and peripheral blood mononuclear cellsare identified by CD56. In some embodiments, M-MDSCs are identified byCD11b and CD14 and peripheral blood mononuclear cells are identified byHLA-DR. In some embodiments, M-MDSCs are identified by CD33 and CD14 andperipheral blood mononuclear cells are identified by CD3. In someembodiments, M-MDSCs are identified by CD33 and CD14 and peripheralblood mononuclear cells are identified by CD14. In some embodiments,M-MDSCs are identified by CD33 and CD14 and peripheral blood mononuclearcells are identified by CD19. In some embodiments, M-MDSCs areidentified by CD33 and CD14 and peripheral blood mononuclear cells areidentified by CD56. In some embodiments, M-MDSCs are identified by CD33and CD14 and peripheral blood mononuclear cells are identified byHLA-DR. In some embodiments, M-MDSCs are identified by CD40 and CD14 andperipheral blood mononuclear cells are identified by CD3. In someembodiments, M-MDSCs are identified by CD40 and CD14 and peripheralblood mononuclear cells are identified by CD14. In some embodiments,M-MDSCs are identified by CD40 and CD14 and peripheral blood mononuclearcells are identified by CD19. In some embodiments, M-MDSCs areidentified by CD40 and CD14 and peripheral blood mononuclear cells areidentified by CD56. In some embodiments, M-MDSCs are identified by CD40and CD14 and peripheral blood mononuclear cells are identified byHLA-DR. In some embodiments, M-MDSCs are identified by CD11b and CD33and CD14 and peripheral blood mononuclear cells are identified by CD3.In some embodiments, M-MDSCs are identified by CD11b and CD33 and CD14and peripheral blood mononuclear cells are identified by CD14. In someembodiments, M-MDSCs are identified by CD11b and CD33 and CD14 andperipheral blood mononuclear cells are identified by CD19. In someembodiments, M-MDSCs are identified by CD11b and CD33 and CD14 andperipheral blood mononuclear cells are identified by CD56. In someembodiments, M-MDSCs are identified by CD11b and CD33 and CD14 andperipheral blood mononuclear cells are identified by HLA-DR. In someembodiments, M-MDSCs are identified by CD11b and CD33 and CD14 andperipheral blood mononuclear cells are identified by CD3, CD14, CD19,CD56, and HLA-DR.

In some embodiments, PMN-MDSCs are identified by CD11b and CD15 andperipheral blood mononuclear cells are identified by CD3. In someembodiments, PMN-MDSCs are identified by CD11b and CD15 and peripheralblood mononuclear cells are identified by CD14. In some embodiments,PMN-MDSCs are identified by CD11b and CD15 and peripheral bloodmononuclear cells are identified by CD19. In some embodiments, PMN-MDSCsare identified by CD11b and CD15 and peripheral blood mononuclear cellsare identified by CD56. In some embodiments, PMN-MDSCs are identified byCD11b and CD15 and peripheral blood mononuclear cells are identified byHLA-DR. In some embodiments, PMN-MDSCs are identified by CD33 and CD15and peripheral blood mononuclear cells are identified by CD3. In someembodiments, PMN-MDSCs are identified by CD33 and CD15 and peripheralblood mononuclear cells are identified by CD14. In some embodiments,PMN-MDSCs are identified by CD33 and CD15 and peripheral bloodmononuclear cells are identified by CD19. In some embodiments, PMN-MDSCsare identified by CD33 and CD15 and peripheral blood mononuclear cellsare identified by CD56. In some embodiments, PMN-MDSCs are identified byCD33 and CD15 and peripheral blood mononuclear cells are identified byHLA-DR. In some embodiments, PMN-MDSCs are identified by CD40 and CD15and peripheral blood mononuclear cells are identified by CD3. In someembodiments, PMN-MDSCs are identified by CD40 and CD15 and peripheralblood mononuclear cells are identified by CD14. In some embodiments,PMN-MDSCs are identified by CD40 and CD15 and peripheral bloodmononuclear cells are identified by CD19. In some embodiments, PMN-MDSCsare identified by CD40 and CD15 and peripheral blood mononuclear cellsare identified by CD56. In some embodiments, PMN-MDSCs are identified byCD40 and CD15 and peripheral blood mononuclear cells are identified byHLA-DR. In some embodiments, PMN-MDSCs are identified by CD11b and CD33and CD15 and Peripheral blood mononuclear cells are identified by CD3.In some embodiments, PMN-MDSCs are identified by CD11b and CD33 and CD15and peripheral blood mononuclear cells are identified by CD14. In someembodiments, PMN-MDSCs are identified by CD11b and CD33 and CD15 andperipheral blood mononuclear cells are identified by CD19. In someembodiments, PMN-MDSCs are identified by CD11b and CD33 and CD15 andperipheral blood mononuclear cells are identified by CD56. In someembodiments, PMN-MDSCs are identified by CD11b and CD33 and CD15 andperipheral blood mononuclear cells are identified by HLA-DR. In someembodiments, PMN-MDSCs are identified by CD11b and CD33 and CD15 andperipheral blood mononuclear cells are identified by CD3, CD14, CD19,CD56, and HLA-DR.

In some embodiments, PMN-MDSCs cells are identified by CD11b an absenceof CD14 on the cell surface and peripheral blood mononuclear cells areidentified by CD3. In some embodiments, PMN-MDSCs are identified byCD11b an absence of CD14 on the cell surface and peripheral bloodmononuclear cells are identified by CD14. In some embodiments, PMN-MDSCsare identified by CD11 an absence of CD14 on the cell and peripheralblood mononuclear cells are identified by CD19. In some embodiments,PMN-MDSCs are identified by CD11b14 an absence of CD14 on the cellsurface and peripheral blood mononuclear cells are identified by CD56.In some embodiments, PMN-MDSCs cells are identified by CD11b an absenceof CD14 on the cell surface and peripheral blood mononuclear cells areidentified by HLA-DR. In some embodiments, PMN-MDSCs cells areidentified by CD33 an absence of CD14 on the cell surface and peripheralblood mononuclear cells are identified by CD3. In some embodiments,PMN-MDSCs are identified by CD33 and an absence of CD14 on the cellsurface and peripheral blood mononuclear cells are identified by CD14.In some embodiments, PMN-MDSCs are identified by CD33 and an absence ofCD14 on the cell surface and peripheral blood mononuclear cells areidentified by CD19. In some embodiments, PMN-MDSCs are identified byCD33 and an absence of CD14 on the cell surface and peripheral bloodmononuclear cells are identified by CD56. In some embodiments, PMN-MDSCsare identified by CD33 and an absence of CD14 on the cell surface andperipheral blood mononuclear cells are identified by HLA-DR. In someembodiments, PMN-MDSCs are identified by CD40 and an absence of CD14 onthe cell surface and peripheral blood mononuclear cells are identifiedby CD3. In some embodiments, PMN-MDSCs are identified by CD40 and anabsence of CD14 on the cell surface and peripheral blood mononuclearcells are identified by CD14. In some embodiments, PMN-MDSCs areidentified by CD40 and an absence of CD14 on the cell surface andperipheral blood mononuclear cells are identified by CD19. In someembodiments, PMN-MDSCs are identified by CD40 and an absence of CD14 onthe cell surface and peripheral blood mononuclear cells are identifiedby CD56. In some embodiments, PMN-MDSCs are identified by CD40 and anabsence of CD14 on the cell surface and peripheral blood mononuclearcells are identified by HLA-DR. In some embodiments, PMN-MDSCs areidentified by CD11b and CD33 and an absence of CD14 on the cell surfaceand peripheral blood mononuclear cells are identified by CD3. In someembodiments, PMN-MDSCs are identified by CD11b and CD33 and an absenceof CD14 on the cell surface and peripheral blood mononuclear cells areidentified by CD14. In some embodiments, PMN-MDSCs are identified byCD11b and CD33 and an absence of CD14 on the cell surface and peripheralblood mononuclear cells are identified by CD19. In some embodiments,PMN-MDSCs are identified by CD11b and CD33 and an absence of CD14 on thecell surface and peripheral blood mononuclear cells are identified byCD56. In some embodiments, PMN-MDSCs are identified by CD11b and CD33and an absence of CD14 on the cell surface and peripheral bloodmononuclear cells are identified by HLA-DR. In some embodiments,PMN-MDSCs are identified by CD11b and CD33 and an absence of CD14 on thecell surface and peripheral blood mononuclear cells are identified byCD3, CD14, CD19, CD56, and HLA-DR.

In some embodiments, CD14-positive and HLA-DR-(lo/negative) cells,CD14-positive cells, and peripheral blood mononuclear cells areidentified by a cell surface marker. Non-limiting examples of cellsurface markers that identify peripheral blood mononuclear cells includeCD3, CD14, CD19, CD56, and HLA-DR.

In some embodiments, CD14-positive and HLA-DR-(lo/negative) cells areidentified by CD14 and a low level or absence of HLA-DR and peripheralblood mononuclear cells are identified by CD3. In some embodiments,CD14-positive and HLA-DR-(lo/negative) cells are identified by CD14 anda low level or absence of HLA-DR and peripheral blood mononuclear cellsare identified by CD14. In some embodiments, CD14-positive andHLA-DR-(lo/negative) cells are identified by CD14 and a low level orabsence of HLA-DR and peripheral blood mononuclear cells are identifiedby CD19. In some embodiments, CD14-positive and HLA-DR-(lo/negative)cells are identified by CD14 and a low level or absence of HLA-DR andperipheral blood mononuclear cells are identified by CD56. In someembodiments, CD14-positive and HLA-DR-(lo/negative) cells are identifiedby CD14 and a low level or absence of HLA-DR and peripheral bloodmononuclear cells are identified by HLA-DR. In some embodiments,CD14-positive and HLA-DR-(lo/negative) cells are identified by CD14 anda low level or absence of HLA-DR and peripheral blood mononuclear cellsare identified by CD3, CD14, CD19, CD56, and HLA-DR. In someembodiments, CD14-positive and HLA-DR-(lo/negative) cells are identifiedby CD14 and a low level or absence of HLA-DR and CD14-positive cells areidentified by CD14.

Additional Therapy

Available additional treatments for triple negative breast cancer thatmay be advantageously employed in combination with the therapiesdisclosed herein include, without limitation, radiation therapy,chemotherapy, antibody therapy, and tyrosine kinase inhibitors asadjuvant therapy.

Radiation therapy is a cancer treatment that uses high-energy x-rays orother types of radiation to kill cancer cells or keep them from growing.Chemotherapy is a cancer treatment that uses drugs to stop the growth ofcancer cells, either by killing the cells or by stopping them fromdividing. When chemotherapy is taken by mouth or injected into a vein ormuscle, the drugs enter the bloodstream and can reach cancer cellsthroughout the body (systemic chemotherapy). When chemotherapy is placeddirectly into the spinal column, an organ, or a body cavity such as theabdomen, the drugs mainly affect cancer cells in those areas (regionalchemotherapy). The way the chemotherapy is given depends on the type andstage of the cancer being treated.

Different chemotherapeutic agents are known in the art for treating lungcancer. Cytoxic agents used for treating lung cancer include carboplatin(for example, Paraplatin®, Paraplat®), cisplatin (for example,Platinol®, Platinol-Aq®), crizotinib (for example Xalkori®), etoposide(for example Toposar®, VePesid®), etoposide Phosphate (for exampleEtopophos®), gemcitabine hydrochloride (for example Gemzar®),gemcitabine-cisplatin, methotrexate (for example Abitrexate®, Folex®,Folex Pfs®, Methotrexate Lpf®, Mexate®, Mexate-Aq®), paclitaxel (forexample Taxol®), pemetrexed Disodium (for example Alimta®), andtopotecan Hydrochloride (for example Hycamtin®)

Different agents are known in the art for treating melanoma, includingaldesleukin (for example Proleukin®), dabrafenib (for exampleTafinlar®), dacarbazine (for example DTIC-Dome®), recombinant InterferonAlfa-2b (for example Intron® A), Ipilimumab (for example Yervoy®),pembrolizumab (for example Keytruda®), Trametinib (for exampleMekinist®), Nivolumab (for example Opdivo®), Peginterferon Alfa-2b (forexample Pegintron®, Sylatron®), vemurafenib (for example Zelboraf®).

Monoclonal antibody therapy is a cancer treatment that uses antibodiesmade in the laboratory, from a single type of immune system cell. Theseantibodies can identify substances on cancer cells or normal substancesthat may help cancer cells grow. The antibodies attach to the substancesand kill the cancer cells, block their growth, or keep them fromspreading. Monoclonal antibodies are given by infusion. They may be usedalone or to carry drugs, toxins, or radioactive material directly tocancer cells. Monoclonal antibodies are also used in combination withchemotherapy as adjuvant therapy.

Additional, illustrative, treatments that may be advantageously combinedwith the compositions and therapies disclosed herein may include,without limitation, administration of agents including, but not limitedto lapatinib, alone or in combination with capecitabine, docetaxel,epirubicin, epothilone A, B or D, goserelin acetate, paclitaxel,pamidronate, bevacizumab, or trastuzumab.

In some embodiments, the additional therapy comprises chemotherapycomprising administering to the subject one or more of doxorubicin,cyclophosphamide, paclitaxel, lapatinib, capecitabine, trastuzumab,bevacizumab, gemcitabine, eribulin, or nab-paclitaxel.

Oral Formulations

Oral formulations containing the active pharmaceutical ingredientsdescribed herein may comprise any conventionally used oral forms,including: tablets, capsules, pills, troches, lozenges, pastilles,cachets, pellets, medicated chewing gum, granules, bulk powders,effervescent or non-effervescent powders or granules, solutions,emulsions, suspensions, solutions, wafers, sprinkles, elixirs, syrups,buccal forms, and oral liquids. Capsules may contain mixtures of theactive compound(s) with inert fillers or diluents such as thepharmaceutically acceptable starches (e.g. corn, potato or tapiocastarch), sugars, artificial sweetening agents, powdered celluloses, suchas crystalline and microcrystalline celluloses, flours, gelatins, gums,etc. Useful tablet formulations may be made by conventional compression,wet granulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. In some embodiments are surfacemodifying agents which include nonionic and anionic surface modifyingagents. For example, surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine. Oral formulations herein mayutilize standard delay or time release formulations to alter theabsorption of the active compound(s). The oral formulation may alsoconsist of administering the active ingredient in water or a fruitjuice, containing appropriate solubilizers or emulsifiers as needed.

Oral Administration

As described herein, the combination therapy described herein can begiven simultaneously or can be given in a staggered regimen, withentinostat being given at a different time during the course ofchemotherapy than the EGFR inhibitor. This time differential may rangefrom several minutes, hours, days, weeks, or longer betweenadministrations of the two compounds. Therefore, the term combinationdoes not necessarily mean administered at the same time or as a unitarydose, but that each of the components are administered during a desiredtreatment period. The agents may also be administered by differentroutes. As is typical for chemotherapeutic regimens, a course ofchemotherapy may be repeated several weeks later, and may follow thesame timeframe for administration of the two compounds, or may bemodified based on patient response.

In other embodiments, the pharmaceutical compositions provided hereinmay be provided in solid, semisolid, or liquid dosage forms for oraladministration. As used herein, oral administration also include buccal,lingual, and sublingual administration. Suitable oral dosage formsinclude, but are not limited to, tablets, capsules, pills, troches,lozenges, pastilles, cachets, pellets, medicated chewing gum, granules,bulk powders, effervescent or non-effervescent powders or granules,solutions, emulsions, suspensions, solutions, wafers, sprinkles,elixirs, and syrups. In addition to the active ingredient(s), thepharmaceutical compositions may contain one or more pharmaceuticallyacceptable carriers or excipients, including, but not limited to,binders, fillers, diluents, disintegrants, wetting agents, lubricants,glidants, coloring agents, dye-migration inhibitors, sweetening agents,and flavoring agents.

Binders or granulators impart cohesiveness to a tablet to ensure thetablet remaining intact after compression. Suitable binders orgranulators include, but are not limited to, starches, such as cornstarch, potato starch, and pre-gelatinized starch (e.g., STARCH 1500);gelatin; sugars, such as sucrose, glucose, dextrose, molasses, andlactose; natural and synthetic gums, such as acacia, alginic acid,alginates, extract of Irish moss, Panwar gum, ghatti gum, mucilage ofisabgol husks, carboxymethylcellulose, methylcellulose,polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powderedtragacanth, and guar gum; celluloses, such as ethyl cellulose, celluloseacetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose, methyl cellulose, hydroxyethylcellulose (HEC),hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC);microcrystalline celluloses, such as AVICEL-PH-101, AVICEL-PH-103,AVICEL RC-581, AVICEL-PH-105 (FMC Corp., Marcus Hook, Pa.); and mixturesthereof. Suitable fillers include, but are not limited to, talc, calciumcarbonate, microcrystalline cellulose, powdered cellulose, dextrates,kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinizedstarch, and mixtures thereof. The binder or filler may be present fromabout 50 to about 99% by weight in the pharmaceutical compositionsprovided herein.

Suitable diluents include, but are not limited to, dicalcium phosphate,calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose,kaolin, mannitol, sodium chloride, dry starch, and powdered sugar.Certain diluents, such as mannitol, lactose, sorbitol, sucrose, andinositol, when present in sufficient quantity, can impart properties tosome compressed tablets that permit disintegration in the mouth bychewing. Such compressed tablets can be used as chewable tablets.

Suitable disintegrants include, but are not limited to, agar; bentonite;celluloses, such as methylcellulose and carboxymethylcellulose; woodproducts; natural sponge; cation-exchange resins; alginic acid; gums,such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses,such as croscarmellose; cross-linked polymers, such as crospovidone;cross-linked starches; calcium carbonate; microcrystalline cellulose,such as sodium starch glycolate; polacrilin potassium; starches, such ascorn starch, potato starch, tapioca starch, and pre-gelatinized starch;clays; aligns; and mixtures thereof. The amount of disintegrant in thepharmaceutical compositions provided herein varies upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. The pharmaceutical compositions provided herein may containfrom about 0.5 to about 15% or from about 1 to about 5% by weight of adisintegrant.

Suitable lubricants include, but are not limited to, calcium stearate;magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol;mannitol; glycols, such as glyceryl behenate and polyethylene glycol(PEG); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetableoil, including peanut oil, cottonseed oil, sunflower oil, sesame oil,olive oil, corn oil, and soybean oil; zinc stearate; ethyl oleate; ethyllaureate; agar; starch; lycopodium; silica or silica gels, such asAEROSIL® 200 (W.R. Grace Co., Baltimore, Md.) and CAB-O-SIL® (Cabot Co.of Boston, Mass.); and mixtures thereof. The pharmaceutical compositionsprovided herein may contain about 0.1 to about 5% by weight of alubricant.

Suitable glidants include colloidal silicon dioxide, CAB-O-SIL® (CabotCo. of Boston, Mass.), and asbestos-free talc. Coloring agents includeany of the approved, certified, water soluble FD&C dyes, and waterinsoluble FD&C dyes suspended on alumina hydrate, and color lakes andmixtures thereof. A color lake is the combination by adsorption of awater-soluble dye to a hydrous oxide of a heavy metal, resulting in aninsoluble form of the dye. Flavoring agents include natural flavorsextracted from plants, such as fruits, and synthetic blends of compoundswhich produce a pleasant taste sensation, such as peppermint and methylsalicylate. Sweetening agents include sucrose, lactose, mannitol,syrups, glycerin, and artificial sweeteners, such as saccharin andaspartame. Suitable emulsifying agents include gelatin, acacia,tragacanth, bentonite, and surfactants, such as polyoxyethylene sorbitanmonooleate (TWEEN® 20), polyoxyethylene sorbitan monooleate 80 (TWEEN®80), and triethanolamine oleate. Suspending and dispersing agentsinclude sodium carboxymethylcellulose, pectin, tragacanth, Veegum,acacia, sodium carboxymethylcellulose, hydroxypropyl methylcellulose,and polyvinylpyrrolidone. Preservatives include glycerin, methyl andpropylparaben, benzoic add, sodium benzoate and alcohol. Wetting agentsinclude propylene glycol monostearate, sorbitan monooleate, diethyleneglycol monolaurate, and polyoxyethylene lauryl ether. Solvents includeglycerin, sorbitol, ethyl alcohol, and syrup. Examples of non-aqueousliquids utilized in emulsions include mineral oil and cottonseed oil.Organic acids include citric and tartaric acid. Sources of carbondioxide include sodium bicarbonate and sodium carbonate.

It should be understood that many carriers and excipients may serveseveral functions, even within the same formulation.

In further embodiments, the pharmaceutical compositions provided hereinmay be provided as compressed tablets, tablet triturates, chewablelozenges, rapidly dissolving tablets, multiple compressed tablets, orenteric-coating tablets, sugar-coated, or film-coated tablets.Enteric-coated tablets are compressed tablets coated with substancesthat resist the action of stomach acid but dissolve or disintegrate inthe intestine, thus protecting the active ingredients from the acidicenvironment of the stomach. Enteric-coatings include, but are notlimited to, fatty acids, fats, phenylsalicylate, waxes, shellac,ammoniated shellac, and cellulose acetate phthalates. Sugar-coatedtablets are compressed tablets surrounded by a sugar coating, which maybe beneficial in covering up objectionable tastes or odors and inprotecting the tablets from oxidation. Film-coated tablets arecompressed tablets that are covered with a thin layer or film of awater-soluble material. Film coatings include, but are not limited to,hydroxyethylcellulose, sodium carboxymethylcellulose, polyethyleneglycol 4000, and cellulose acetate phthalate. Film coating imparts thesame general characteristics as sugar coating. Multiple compressedtablets are compressed tablets made by more than one compression cycle,including layered tablets, and press-coated or dry-coated tablets.

The tablet dosage forms may be prepared from the active ingredient inpowdered, crystalline, or granular forms, alone or in combination withone or more carriers or excipients described herein, including binders,disintegrants, controlled-release polymers, lubricants, diluents, orcolorants. Flavoring and sweetening agents are especially useful in theformation of chewable tablets and lozenges.

The pharmaceutical compositions provided herein may be provided as softor hard capsules, which can be made from gelatin, methylcellulose,starch, or calcium alginate. The hard gelatin capsule, also known as thedry-filled capsule (DFC), consists of two sections, one slipping overthe other, thus completely enclosing the active ingredient. The softelastic capsule (SEC) is a soft, globular shell, such as a gelatinshell, which is plasticized by the addition of glycerin, sorbitol, or asimilar polyol. The soft gelatin shells may contain a preservative toprevent the growth of microorganisms. Suitable preservatives are thoseas described herein, including methyl- and propyl-parabens, and sorbicacid. The liquid, semisolid, and solid dosage forms provided herein maybe encapsulated in a capsule. Suitable liquid and semisolid dosage formsinclude solutions and suspensions in propylene carbonate, vegetableoils, or triglycerides. Capsules containing such solutions can beprepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and4,410,545. The capsules may also be coated as known by those of skill inthe art in order to modify or sustain dissolution of the activeingredient.

In other embodiments, the pharmaceutical compositions provided hereinmay be provided in liquid and semisolid dosage forms, includingemulsions, solutions, suspensions, elixirs, and syrups. An emulsion is atwo-phase system, in which one liquid is dispersed in the form of smallglobules throughout another liquid, which can be oil-in-water orwater-in-oil. Emulsions may include a pharmaceutically acceptablenon-aqueous liquids or solvent, emulsifying agent, and preservative.Suspensions may include a pharmaceutically acceptable suspending agentand preservative. Aqueous alcoholic solutions may include apharmaceutically acceptable acetal, such as a di(lower alkyl) acetal ofa lower alkyl aldehyde (the term “lower” means an alkyl having between 1and 6 carbon atoms), e.g., acetaldehyde diethyl acetal; and awater-miscible solvent having one or more hydroxyl groups, such aspropylene glycol and ethanol. Elixirs are clear, sweetened, andhydroalcoholic solutions. Syrups are concentrated aqueous solutions of asugar, for example, sucrose, and may also contain a preservative. For aliquid dosage form, for example, a solution in a polyethylene glycol maybe diluted with a sufficient quantity of a pharmaceutically acceptableliquid carrier, e.g., water, to be measured conveniently foradministration.

Other useful liquid and semisolid dosage forms include, but are notlimited to, those containing the active ingredient(s) provided herein,and a dialkylated mono- or poly-alkylene glycol, including,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 referto the approximate average molecular weight of the polyethylene glycol.These formulations may further comprise one or more antioxidants, suchas butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA),propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine,lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoricacid, bisulfate, sodium metabisulfite, thiodipropionic acid and itsesters, and dithiocarbamates.

The pharmaceutical compositions provided herein for oral administrationmay be also provided in the forms of liposomes, micelles, microspheres,or nanosystems. Miccellar dosage forms can be prepared as described inU.S. Pat. No. 6,350,458.

In other embodiments, the pharmaceutical compositions provided hereinmay be provided as non-effervescent or effervescent, granules andpowders, to be reconstituted into a liquid dosage form. Pharmaceuticallyacceptable carriers and excipients used in the non-effervescent granulesor powders may include diluents, sweeteners, and wetting agents.Pharmaceutically acceptable carriers and excipients used in theeffervescent granules or powders may include organic acids and a sourceof carbon dioxide.

Coloring and flavoring agents can be used in all of the above dosageforms.

The pharmaceutical compositions provided herein may be formulated asimmediate or modified release dosage forms, including delayed-,sustained, pulsed-, controlled, targeted-, and programmed-release forms.

In further embodiments, the pharmaceutical compositions provided hereinmay be co-formulated with other active ingredients which do not impairthe desired therapeutic action, or with substances that supplement thedesired action.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The disclosurehaving now been described by way of written description, those of skillin the art will recognize that the disclosure can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1 Modulation of Myeloid Derived Suppressor Cells byEntinostat

Blood samples were obtained from a set of 49 patients enrolled in theENCORE 301 clinical trial, all of whom had advanced hormonereceptor-positive breast cancer and had progressed on a non-steroidalaromatase inhibitor. 27 patients received exemestane and entinostat(“EE”) and 22 received exemestane and placebo (“EP”). Blood samples wereobtained on cycle 1 day 1 (C1D1; pre-treatment), C1D2, C1D8, and C1D15for biomarker analyses. Of these patient samples, 34 (20 EE and 14 EP)were analyzed for circulating immune subsets. Cell populations werebased on the following surface markers, in which “X−” means negative formarker X, “X+” means positive for marker X, “Xlow/−” means low levels toabsence of marker X, and “Xhi” means high levels of expression of markerX:

Myeloid derived suppressor cell: CD3−, CD19−, CD56−, HLA-DR−, CD11b+,CD33+.

PMN-MDSC: CD14−, CD11b+, CD33+.

M-MDSC: CD3−, CD19−, CD56−, HLA-DR−, CD11b+, CD33+, CD14+.

Immature MDSC: CD3−, CD19−, CD56−, HLA-DR−, CD11b+, CD33+, CD14−.

CD8+ T-cell: CD4−, CD8+, Foxp3−.

CD4+ T-cell: CD8−, CD4+, Foxp3−.

T_(regs): CD4+, CD8−, CD25hi, Foxp3+.

Monocytes were analyzed for three populations: (1) CD14+; (2) CD14+,HLA-DRhi; and (3) CD14+, HLA-DRlow/−. PD-1, CTLA-4, and TIM-3 weremeasured on CD8+ T-cells, CD4+ T-cells, and T_(regs), and CD40 wasmeasured on myeloid derived suppressor cells.

All comparisons were performed between the C1D1 and the C1D15 timepoints in the EE and EP treatment groups. For PMN-MDSCs, EE treatmentled to a change of -14.67%, while EP treatment led to a change of+20.56%. For M-MDSCs, EE treatment led to a change of −62.3%, while EPtreatment led to a change of +1.97%. For immature MDSCs, EE treatmentled to a change of −20.9%, while EP treatment led to a change of −15.0%.In myeloid derived suppressor cells, M-MDSCs, PMN-MDSCs, and immaturemyeloid derived suppressor cells, CD40 levels were reduced in the EEtreatment group compared to the EP treatment group. For HLA-DR+monocytes, EE treatment led to a change of +34.1%, while EP treatmentled to a change of −11.38%. On monocytes, HLA-DR levels were +16.3% inthe EE treatment group and −4.7% in the EP treatment group.

Example 2 Modulation of CD14⁺-HLA-DR(Lo/Negative) Myeloid DerivedSuppressor Cells by Entinostat Combination Therapy

Entinostat, a class I HDAC inhibitor (HDACi), has shown promisingactivity in ENCORE 301, a randomized, placebo-controlled, phase II trialof entinostat plus exemestane (“EE”) vs. exemestane plus placebo (“EP”)in advanced hormone receptor positive breast cancer that had progressedon non-steroidal aromatase inhibitors. ENCORE 301 met the primaryprogression free survival end-point and showed a median 8.3 monthimprovement in the overall survival (OS) endpoint for the EE arm. Basedon those results entinostat was granted breakthrough therapy designationand a Phase 3 trial, E2112 comparing EE to EP is currently enrolling.Emerging preclinical work suggests that entinostat has immunomodulatoryeffects on immune suppressor cells including regulatory T cells (Tregs)and myeloid derived suppressor cells (MDSCs) and can eradicate modestlyimmunogenic mouse tumors in combination with immune checkpoint blockadeagents. This activity was shown to be mediated via reduction of MSDCs(Kim et al., PNAS 2014,111:11774-9). These results may be explained byentinostat's selective targeting of those class 1 HDAC enzymes whichhave been shown to play a role in the differentiation and activation ofTregs (Shen et'al. PLoS One 2012, 7:e30815; Wang et al. JCI 2015,125:1111-1123) and MDSCs (Youn et al. Nat. Immunol 2013,14:211-220)_([OL1]).

Based on these data, an analysis was conducted of immune subsets inblood samples from ENCORE 301 breast cancer patients.

Blood was collected from a subset of 49 patients (27 EE and 22 EP)representative of the 130 patients enrolled in ENCORE 301 on cycle 1 day1 (C1D1; pretreatment), C1D2, C1D8, and C1D15 for biomarker analyses. Ofthese 49, C1D1 and C1D15 samples were available from 34 patients (20 EEand 14 EP) for further analysis of circulating immune subsets. Theclinical outcome for Progression Free Survival (PFS) was found to be 4.9months for EE (Entinostat +Exemestane) patients , 1.8 months for(Exemestane and+Placebo) with a hazard ratio (HR) of 0.62 months andOverall Survival (OS) was found to be 28.1 months for EE, 20.3 monthsfor EP, with a HR of 0.62 months, in the 34 patients as well asbaselined demographics was consistent with the intent-to-treatpopulation.

The percent change in subsets at C1D15 vs. baseline was assessed basedon the following surface markers: Lin-MDSC (lin;CD3,CD19,CD56)-HLA-DR-CD11b+CD33+), granulocytic MDSC(CD14-CD11b+CD33+), monocytic MDSC (Lin-HLA-DR-CD11b+CD33+CD14+),immature MDSC (Lin-HLA-DR-CD11b+CD33+CD14-), CD8+T-cells (CD4-CD8+),Foxp3-CD4+T-cells (CD8-CD4+Foxp3−), and Tregs (CD4+CD8-CD25hiFoxp3+).Monocytes were analyzed for three populations: CD14+, CD14+HLA-DRhi, andCD14+HLA-DR-low/negative. In addition, PD-1, CTLA-4, and TIM-3 weremeasured on T-cell subsets, and CD40 was measured on MDSCs.

In line with preclinical data, a reduction in granulocytic MDSC (-14.67%vs. +20.56%, P=0.03) and monocytic MDSC (−62.3% vs. +1.97%, P=0.002) wasobserved in EE. Interestingly, CD40, a costimulatory receptor requiredfor MDSC-mediated immune suppression was also down-regulated in all MDSCsubsets. Entinostat did not significantly impact the ratio of CD8+T-cells to CD4+ T-cells or alter expression of CTLA-4, PD-1, or TIM-3 onT-cell subsets. Reduced expression of HLA-DR on monocytes has beenassociated with poor prognosis in cancer. Consistent withentinostat-mediated immunomodulatory effects, an increase in the numberof HLA-DR+monocytes (34.1% vs. −11.38%, P=0.0004) and level of HLA-DRexpression on monocytes (16.3% vs. −4.7%; P=0.015) was observed. Nocorrelation was identified between the C1D15 immune cell changes andclinical outcome in this subset of patients.

Results are shown in Table 1 below.

TABLE 1 Effect of EE vs EP treatment on surface markers EP (n = 14;change from EE (n = 20; change from baseline to C1D15 (%)) baseline toC1D15 (%)) p-value Lab Test Median (Min, Mean Median (Min, (EE vs NameMean (SD) Max) (SD) Max) EP) CD14+ −0.58 (22.73) 1.90 (−38.67,  15.71(28.49)  13.12 (−22.76, 0.16 42.09) 65.47) CD14 + HLA- −8.63 (17.77)−11.38 (−37.35,   41.76 (44.28)  34.08 (−25.35, 0.0004 DR^(HI) 25.37)123.84) CD14 + HLA- 13.45 (43.11) 1.55 (−41.10,  3.56 (42.07)  −3.46(−47.33, 0.45 DR^(LO/NEG) 93.38) 66.14) HLA-DR −6.23 (18.77) −4.74(−32.46,   23.20 (36.69)  16.26 (−36.75, 0.02 expression 29.94) 89.30)on CD14+ g-MDSCs 20.56 (68.45) 3.82 (−72.47, −14.67 (65.78) −34.53(−77.75, 0.03 224.31) 220.55) CD40 on 16.52 (32.07) 10.52 (−27.59,  −0.35 (30.88)  −0.72 (−47.74, 0.22 g-MDSCs 75.76) 50.00) m-MDSCs 28.24(86.60) 1.97 (−55.05, −44.94 (50.22) −62.33 (−92.81, 0.002 241.46)85.71) CD40 on  3.37 (25.69) 1.57 (−45.55, −16.75 (21.37) −17.18(−61.05, 0.011 m-MDSCs 73.91) 25.29) Lin-MDSCs  1.28 (83.31) −22.11(−58.15,  −13.26 (58.66) −29.03 (−83.61, 0.611 274.45) 120.51) CD40 on14.85 (34.92) 3.37 (−26.61, −16.67 (26.47) −15.38 (−71.05, 0.02Lin-MDSCs 97.78) 21.82) immature  4.92 (94.50) −20.94 (−68.27,   18.75(130.14) −14.95 (−89.23, 0.93 MDSCs 306.60) 467.57) CD40 on 18.64(35.15) 9.00 (−32.58, −11.61 (21.90)  −8.51 (−49.30, 0.007 immature86.36) 31.25) MDSCs

Further, as seen in FIG. 1 , for HLA-DR⁺ monocytes, EE treatment led toa change of +34.1%, while EP treatment led to a change of −11.38%. Onmonocytes, HLA-DR levels were +16.3% in the EE treatment group and −4.7%in the EP treatment group. More data are shown in FIGS. 2-5 . Themethods and results are also described in Tomita et al., “The interplayof epigenetic therapy and immunity in locally recurrent or metastaticestrogen receptor-positive breast cancer: Correlative analysis of ENCORE301, a randomized, placebo-controlled phase II trial of exemestane withor without entinostat”, ONCOIMMUNOLOGY, Taylor and Francis Online, 2016,the entire contents of which are hereby incorporated by reference in itsentireties.

In summary, data from blood samples obtained from ER+ breast cancerpatients treated with entinostat combined with exemestane in ENCORE 301provided the first evidence of HDACi-mediated reduction ofimmunosuppressive MDSCs and increased immunocompetent CD14+HLA-DR^(HI)monocytes in patients. These findings may in part explain the improvedOS seen with EE in ENCORE 301 and provide strong rationale for plannedcombination studies of entinostat with immune checkpoint blockade.

The reduction in MDSCs in entinostat treated patients was consistentwith recently published preclinical work (Kim et al PNAS 2014)demonstrating entinostat's ability to enhance the anti-tumor activity ofimmune checkpoint inhibitors through reduction of MDSCs. A phase 1b/2trial of entinostat in combination with pembrolizumab in NSCLC andmelanoma has been initiated to determine the safety and clinical benefitof the combination.

A phase 1b/2 trial of entinostat in combination with atezolizumab inTNBC is planned.

Additional preclinical and clinical studies are ongoing to furtherexplore entinostat's immunomodulatory activity.

Example 3 Patient Selection for Entinostat Combination Therapy withExemestane

To select a patient for treatment with entinostat in combination withexemestane, a peripheral blood sample is taken from the patient. Thepatient is a post-menopausal woman diagnosed with metastatichormone-receptor positive breast cancer who has progressed duringtreatment with a non-steroidal aromatase inhibitor. The 5 milliliter(mL) peripheral blood sample is taken into EDTA collection tubes, whichare rapidly cooled on ice. Blood samples are transferred to a conicaltube and diluted with 15 mL of red blood cell lysis buffer and incubatedat room temperature for 10 minutes. Red blood cell lysis is quenched bydilution with 30 mL phosphate-buffered saline (PBS). The cell suspensionis centrifuged 5 minutes at 400×g at 4° C. and the supernatant isdiscarded. The pellet is resuspended in 5 mL of PBS and transferred to anew conical tube. The resuspended sample is underlaid with 5 mL Ficoll®.Cells are centrifuged for 20 minutes 400×g with the brake turned off onthe centrifuge. Nucleated cells are harvested at the interface of thePBS and Ficoll® layers into a fresh conical tube.

An equal volume to the original blood draw is added to the pellet in thefresh conical tube to wash the cells. The cell suspension is centrifuged5 minutes at 400×g at 4° C. and the supernatant is discarded. Cells areresuspended in an equal volume to the original blood draw in PBS with 1%bovine serum albumin and 0.5% EDTA (staining buffer). Viable cells arethen counted using a hemacytometer. The cell suspension is centrifuged 5minutes at 400×g at 4° C., the supernatant is discarded, and the cellsare resuspended in Staining Buffer to a cell concentration of about 10⁷cells per mL and 1 mL aliquots are transferred to new tubes.

In a first study, the following antibodies are added to the resuspendedcells: FITC-conjugated anti-CD11b; PE-conjugated anti-CD33;PerCP-Cy™5.5-conjugated anti-CD14; and APC-conjugated anti-CD3,anti-CD19, anti-CD56, and anti-HLA-DR. In a second study, the followingantibodies are added to the resuspended cells: FITC-conjugatedanti-CD14; PE-conjugated anti-HLA-DR; and APC-conjugated anti-CD3,anti-CD19, and anti-CD56. In a third study, the following antibodies areadded to the resuspended cells: FITC-conjugated anti-CD14 andPE-conjugated anti-HLA-DR. Control samples include unstained cells andstained cells in which one of each of the set of fluorochrome antibodiesis left out. Cells are covered to minimize light exposure and left atroom temperature for 20 minutes. Stained cells are washed twice instaining buffer by centrifugation for 5 minutes at 400×g at 4° C.,discarding of the supernatant, and resuspension in an equal volume ofstaining buffer. Stained cells are then transferred to polypropylenetubes for use on the flow cytometer.

Flow cytometry is performed on a Cytomics FC 500 flow cytometer, whichautomates tube-based acquisition of flow cytometry data. Afterperformance of the automated run, samples are corrected both forbackground fluorescence (using the unstained sample) and fluorochromecompensation (using the individually left out fluorochrome samples). Inthe first study, antibody-cell complexes are then calculated for myeloidderived suppressor cells, identified by CD11b and CD33 positivity andCD3, CD19, CD56, HLA-DR negativity, and Peripheral blood mononuclearcells, identified by CD3, CD14, CD19, CD56, and HLA-DR positivity andCD11b and CD33 negativity. These values for the antibody-cell complexesare used to calculate the ratio of myeloid derived suppressor cells toperipheral blood mononuclear cells. In this setting, if the patient hasa ratio of between 1:100 and 1:1, indicating the presence of elevatednumbers of myeloid derived suppressor cells, the patient is selected forcombination therapy with entinostat and exemestane. In the second study,CD14-positive and HLA-DR-lo/negative cells are identified by CD14positivity, HLA-DR low expression or negativity, and CD3, CD19, and CD56negativity. Peripheral blood mononuclear cells, identified by CD3, CD19,CD56, and HLA-DR positivity. These values are used to calculate theratio of CD14-positive and HLA-DR-lo/negative cells to peripheral bloodmononuclear cells. In this setting, if the patient has a ratio ofbetween 1:200 and 1:1, indicating the presence of elevated numbers ofCD14-positive and HLA-DR-lo/negative cells, the patient is selected forcombination therapy with entinostat and exemestane. In the third study,CD14-positive and HLA-DR-lo/negative cells are identified by CD14positivity and HLA-DR low expression or negativity. CD14-positive cellsare identified by CD14 positivity independent of HLA-DR expression.These values are used to calculate the ratio of CD14-positive andHLA-DR-lo/negative cells to CD14-positive cells. In this setting, if thepatient has a ratio of between 1:100 and 99:1, indicating the presenceof elevated numbers of CD14-positive and HLA-DR-lo/negative cells, thepatient is selected for combination therapy with entinostat andexemestane.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1.-2. (canceled)
 3. A method of providing a prognosis for cancer in apatient comprising: obtaining a peripheral blood sample from thepatient, wherein the patient is diagnosed with a cancer; measuring thenumber of myeloid derived suppressor cells and peripheral bloodmononuclear cells in the peripheral blood sample, wherein the methodfurther comprises administering a combination therapy comprisingentinostat and a second therapeutic agent to the patient, if the ratioof myeloid derived suppressor cells to peripheral blood mononuclearcells is between 1:200 and 1:4.
 4. The method of claim 3, wherein theperipheral blood sample is treated with an anticoagulant.
 5. (canceled)6. The method of claim 3, wherein measuring the number of myeloidderived suppressor cells and the peripheral blood mononuclear cells(PBMC) is performed by flow cytometry or by cytospin.
 7. (canceled) 8.The method of claim 3, wherein the number of myeloid derived suppressorcells is identified by a cell surface marker.
 9. The method of claim 8,wherein the cell surface marker is at least one of CD11b, CD33, andCD40.
 10. The method of claim 3, wherein the number of peripheral bloodmononuclear cells is identified by a cell surface marker.
 11. (canceled)12. The method of claim 3, wherein the myeloid derived suppressor cellis a polymorphonuclear-MDSC or a monocytic-MDSC.
 13. The method of claim12, wherein the polymorphonuclear-MDSC or the monocytic-MDSC isidentified by a cell surface marker. 14.-17. (canceled)
 18. The methodof claim 3, wherein the ratio is selected from the group consisting ofbetween 1:100 and 1:4, between 1:50 and 1:4, between 1:20 and 1:4,between 1:10 and 1:4, between 1:10 and 1:4, and between 1:5 and 1:4.19.-24. (canceled)
 25. The method of claim 3, wherein the entinostat isadministered weekly or every two weeks.
 26. (canceled)
 27. The method ofclaim 3, wherein the second therapeutic agent is an anti-PD-1 antibody.28.-35. (canceled)
 36. The method of claim 3, wherein the secondtherapeutic agent is exemestane. 37.-42. (canceled)
 43. The method ofclaim 3, wherein the first and second binding agents each independentlybind a cell surface marker. 44.-45. (canceled)
 46. A method of selectinga patient for combination therapy comprising entinostat and a secondtherapeutic agent comprising: obtaining a peripheral blood sample fromthe patient, wherein the patient is diagnosed with a cancer; measuringthe number of cells in the peripheral blood sample which areCD14-positive and HLA-DR-(lo/negative); measuring the number of cells inthe peripheral blood sample which are CD-14 positive; and administeringthe combination therapy if the ratio of the CD14-positive andHLA-DR-(lo/negative) cells to CD-14 positive cells is between 1:100 and99:1.
 47. (canceled)
 48. A method of providing a prognosis for cancer ina patient comprising: obtaining a peripheral blood sample from thepatient, wherein the patient is diagnosed with a cancer; measuring thenumber of cells in the peripheral blood sample which are CD14-positiveand HLA-DR-(lo/negative); measuring the number of cells in theperipheral blood sample which are CD-14 positive, wherein the methodfurther comprises, administering a combination therapy comprisingentinostat and a second therapeutic agent to the patient, if the ratioof CD14-positive and HLA-DR-(lo/negative) cells to CD-14 positive cellsis between 1:100 and 99:1.
 49. (canceled)
 50. The method of claim 48,wherein the peripheral blood sample is treated with an anticoagulant.51. (canceled)
 52. The method of claim 48, wherein measuring number ofcells in the peripheral blood sample which are CD14-positive andHLA-DR-(lo/negative) is performed by flow cytometry. 53.-64. (canceled)65. The method of claim 48, wherein the entinostat is administeredweekly or every two weeks.
 66. The method of claim 48, wherein theentinostat is administered every two weeks.
 67. The method of claim 48,wherein the second therapeutic agent is an anti-PD-1 antibody or ananti-PD-L1 antibody. 68.-81. (canceled)